Peptides that selectively home to heart vasculature and related conjugates and methods

ABSTRACT

The present invention provides a variety of isolated peptides and peptidomimetics, which can be useful, for example, in constructing the conjugates of the invention or, where the peptide itself has biological activity, in unconjugated form as a therapeutic for treating any of a variety of cardiovascular diseases as described below. Thus, the present invention provides an isolated peptide or peptidomimetic which has a length of less than 60 residues and includes the amino acid sequence CRPPR (SEQ ID NO: 1) or a peptidomimetic thereof. The invention further provides an isolated peptide or peptidomimetic which has a length of less than 60 residues and includes the amino acid sequence CARPAR (SEQ ID NO: 5) or a peptidomimetic thereof, or amino acid sequence CPKRPR (SEQ ID NO: 6) or a peptidomimetic thereof.

This is a continuation of U.S. application Ser. No. 11/222,370, filedSep. 7, 2005, which claims the benefit of U.S. provisional applicationSer. No. 60/714,515, filed Sep. 7, 2004, which was converted from U.S.application Ser. No. 10/936,027, filed Sep. 7, 2004, each of which theentire contents is incorporated herein by reference.

This invention was made with government support under CA 82713, CA30199, NCI training grant T32 CA77109-05 and DOD training grant DAMD17-02-0309. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularmedicine and drug and gene delivery and, more specifically, tocardiovascular disease and novel compositions for treatingcardiovascular disease.

2. Background Information

Atherosclerosis and its sequelae constitute the most common andimportant cause of disease and death in the western world, accountingfor 50% of all deaths in the west. As described further below,atherosclerosis is a disorder which results from the thickening andinelasticity of vessel walls or arteries as a consequence of atheroma.

In normal hearts, vessel walls are composed of an endothelial celllining that is tightly juxtaposed to a medial layer of vascular smoothmuscle cells with an over layer of connective tissue. The endothelialcell lining is ideally situated at the interface between the blood andthe vessel wall to transduce signals, with endothelial cells controllingthe homeostatic balance of the vessel through the production of factorsregulating processes such as vessel tone, coagulation state, cellgrowth, cell death, and leukocyte trafficking. Vascular smooth musclecells maintain the contractile tone of the blood vessel in response tovasoactive agents, and release cytokines and other growth factors. Inconjunction with fibroblasts, the smooth muscle cells produceextracellular matrix proteins and proteases that determine vesselstructure. Occlusive vascular disease, the most common form of which isatherosclerosis, is characterized by an abnormal accumulation of lipid,inflammatory cells, vascular smooth muscle cells and extracellularmatrix proteins within the intimal space between the endothelial liningand the medial layer (plaque formation).

Therapies for atherosclerosis generally prevent, arrest or reverse theprocess of plaque formation or stimulate the formation of new bloodvessels. It is rare, however, that a drug or other agent for treatmentof atherosclerosis targets only coronary vessels. More commonly,systemic administration results in undesirable side effects due, forexample, to generalized toxic effects throughout the entire body. Forexample, vascular endothelial growth factor (VEGF), a key regulator ofangiogenesis that stimulates endothelial cell proliferation, promotesthe formation of new vessels, thereby increasing blood flow to ischemictissue and relieving vascular disease. However, VEGF also can promotenonspecific mitogenesis and potentiate angiogenesis-driven diseases suchas diabetic retinopathy, and certain tumors. Similarly, systemicadministration of the angiogenic stimulator fibroblast growth factor cancause severe side effects due to its lack of specificity for cardiactissue. Unfortunately, such side effects, which include repetitiveepisodes of hypertension, limit the utility of existing pro-angiogenictherapies for treatment of atherosclerosis.

As the cells which make up the internal lining of blood vessels,endothelial cells are the first cell type encountered by a circulatingdrug or other substance. Endothelial cells therefore provide a targetfor selectively directing a therapeutic substance to cardiac tissue.Such selective targeting of a drug or other therapeutic substance toheart vasculature would reduce or eliminate the risk of unwanted sideeffects such as systemic toxicity or malignant transformation. Selectivetargeting of a therapeutic substance to heart vasculature also wouldeffect a high local concentration of the substance, thereby reducing thedosage required for effective treatment.

Thus, a need exists to identify molecules that selectively bind to heartvasculature in vivo. Such molecules would be particularly useful forselectively targeting therapeutic agents to the heart for treatment ofcardiopathies and cardiovascular diseases such as atherosclerosis. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

The present invention provides a variety of isolated peptides andpeptidomimetics, which can be useful, for example, in constructing theconjugates of the invention or, where the peptide itself has biologicalactivity, in unconjugated form as a therapeutic for treating any of avariety of cardiovascular diseases as described below. Thus, the presentinvention provides an isolated peptide or peptidomimetic which has alength of less than 60 residues and includes the amino acid sequenceCRPPR (SEQ ID NO: 1) or a peptidomimetic thereof. The invention furtherprovides an isolated peptide or peptidomimetic which has a length ofless than 60 residues and includes the amino acid sequence CARPAR (SEQID NO: 5) or a peptidomimetic thereof, or amino acid sequence CPKRPR(SEQ ID NO: 6) or a peptidomimetic thereof.

The invention further provides an isolated peptide or peptidomimeticwhich includes the amino acid sequence GRKSKTV (SEQ ID NO: 14) or apeptidomimetic thereof. In one embodiment, the invention provides anisolated peptide or peptidomimetic which includes the amino acidsequence CXGRKSKTVZC (SEQ ID NO: 15) or a peptidomimetic thereof, whereX=0 to 20 independently selected residues and Z=0 to 20 independentlyselected residues.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 150 residues and includesthe amino acid sequence CARPAR (SEQ ID NO: 5) or a peptidomimeticthereof.

Also provided herein is an isolated peptide or peptidomimetic which hasa length of less than 50 residues and includes the amino acid sequenceCPKRPR (SEQ ID NO: 6) or a peptidomimetic thereof.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence CKRAVR (SEQ ID NO: 7) or a peptidomimeticthereof.

Further provided by the invention is an isolated peptide orpeptidomimetic which includes the amino acid sequence RNSWKPN (SEQ IDNO: 16) or a peptidomimetic thereof. In one embodiment, the inventionprovides an isolated peptide or peptidomimetic which includes the aminoacid sequence CXRNSWKPNZC (SEQ ID NO: 17) or a peptidomimetic thereof,where X=0 to 20 independently selected residues and Z=0 to 20independently selected residues.

Further provided herein is an isolated peptide or peptidomimetic whichincludes the amino acid sequence RGSSS (SEQ ID NO: 9) or apeptidomimetic thereof.

The present invention also provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence RSTRANP (SEQ ID NO: 18) or a peptidomimeticthereof. In one embodiment, an isolated peptide or peptidomimetic of theinvention includes the amino acid sequence CXRSTRANPZC (SEQ ID NO: 19)or a peptidomimetic thereof, where X=0 to 20 independently selectedresidues and Z=0 to 20 independently selected residues.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence PKTRRVP (SEQ ID NO: 20) or a peptidomimeticthereof. In one embodiment, an isolated peptide or peptidomimeticincludes the amino acid sequence CXPKTRRVPZC (SEQ ID NO: 21) or apeptidomimetic thereof, where X=0 to 20 independently selected residuesand Z=0 to 20 independently selected residues.

Further provided herein is an isolated peptide or peptidomimetic whichhas a length of less than 400 residues and includes the amino acidsequence SGMARTK (SEQ ID NO: 22) or a peptidomimetic thereof. In oneembodiment, the invention provides an isolated peptide or peptidomimeticwhich includes the amino acid sequence CXSGMARTKZC (SEQ ID NO: 23) or apeptidomimetic thereof, where X=0 to 20 independently selected residuesand Z=0 to 20 independently selected residues.

Also provided herein is a method of isolating one or more homingmolecules that selectively homes to heart vasculature by contactingHLP/CRIP2, or a fragment thereof, with a library of molecules underconditions suitable for specific binding of a molecule to HLP/CRIP2;assaying for specific binding; and separating one or moreHLP/CRIP2-binding molecules from the library, thereby isolating one ormore homing molecules that selectively homes to heart vasculature andspecifically binds HLP/CRIP2.

Further provided by the present invention is a method of isolating oneor more homing molecules that selectively homes to heart vasculature bycontacting receptor clone 9, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto receptor clone 9; assaying for specific binding; and separating oneor more receptor clone 9-binding molecules from the library, therebyisolating one or more homing molecules that selectively homes to heartvasculature and specifically binds receptor clone 9.

The present invention also provides a method of isolating one or morehoming molecules that selectively homes to heart vasculature bycontacting Sigirr/TIR8, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto Sigirr/TIR8; assaying for specific binding; and separating one ormore Sigirr/TIR8-binding molecules from the library, thereby isolatingone or more homing molecules that selectively homes to heart vasculatureand specifically binds Sigirr/TIR8.

Further provided herein is a method of isolating one or more homingmolecules that selectively homes to heart vasculature by contactingMpcII-3-related protein, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto MpcII-3-related protein; assaying for specific binding; andseparating one or more MpcII-3-related protein-binding molecules fromthe library, thereby isolating one or more homing molecules thatselectively homes to heart vasculature and specifically bindsMpcII-3-related protein.

The present invention further provides a method of isolating one or morehoming molecules that selectively homes to heart vasculature bycontacting bc10, or a fragment thereof, with a library of moleculesunder conditions suitable for specific binding of a molecule to bc10;assaying for specific binding; and separating one or more bc10-bindingmolecules from the library, thereby isolating one or more homingmolecules that selectively homes to heart vasculature and specificallybinds bc10.

Also provided herein is a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to an antibody, or antigen-bindingfragment thereof, that selectively homes to heart vasculature and thatspecifically binds HLP/CRIP2 (SEQ ID NO: 25), thereby directing themoiety to heart vasculature.

The present invention further provides a method of directing a moiety toheart vasculature in a subject in which a conjugate is administered to asubject, the conjugating containing a moiety linked to an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and specifically binds SEQ ID NO:27, thereby directing themoiety to heart vasculature.

Also provided herein is a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to an antibody, or antigen-bindingfragment thereof, that selectively homes to heart vasculature and thatspecifically binds Sigirr/TIR8 (SEQ ID NO: 29), thereby directing themoiety to heart vasculature.

Further provided herein is a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to an antibody, or antigen-bindingfragment thereof, that selectively homes to heart vasculature and thatspecifically binds SEQ ID NO: 33, thereby directing the moiety to heartvasculature.

The present invention additionally provides a method of directing amoiety to heart vasculature in a subject by administering to the subjecta conjugate which contains moiety linked to an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and that specifically binds bc10 (SEQ ID NO: 35), therebydirecting the moiety to heart vasculature.

The present invention also provides a conjugate containing a therapeuticagent linked to a homing molecule that selectively homes to heartvasculature and specifically binds cysteine-rich protein 2 (HLP/CRIP2;SEQ ID NO: 25). Also provided herein is a method of directing a moietyto heart vasculature in a subject by administering to the subject aconjugate containing a moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically binds HLP/CRIP2(SEQ ID NO: 25), thereby directing the moiety to heart vasculature.

The present invention further provides a conjugate containing atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature and specifically binds receptor clone 9 (SEQ ID NO:27). Also provided herein is a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatecontaining a moiety linked to a homing molecule that selectively homesto heart vasculature and specifically binds receptor clone 9 (SEQ ID NO:27), thereby directing the moiety to heart vasculature.

Further provided herein is a conjugate containing a therapeutic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds single Ig IL-2 receptor related protein(Sigirr/TIR8; SEQ ID NO: 29). Such a conjugate can be useful, forexample, in a method of the invention for directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to a homing molecule that selectivelyhomes to heart vasculature and specifically binds Sigirr/TIR8 (SEQ IDNO: 29), thereby directing the moiety to heart vasculature.

The present invention also provides a conjugate which contains atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature and specifically binds SEQ ID NO: 33, a proteinproduct having putative similarity to integral membrane protein CII-3(MpcII-3). The invention additionally provides methods of directing amoiety to heart vasculature in a subject by administering to the subjecta conjugate containing a moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically binds SEQ ID NO:33, thereby directing the moiety to heart vasculature.

Also provided herein is a conjugate containing a therapeutic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds murine bladder cancer-associated protein homolog(bc10; SEQ ID NO: 35). The invention additionally provides methods ofdirecting a moiety to heart vasculature in a subject by administering tothe subject a conjugate containing a moiety linked to a homing moleculethat selectively homes to heart vasculature and specifically binds bc10(SEQ ID NO: 35), thereby directing the moiety to heart vasculature.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows identification of homing peptide/receptor pairs by ex vivoand in vivo phage display combined with bacterial two-hybrid analysis.(A) The principle of parallel homing peptide isolation and receptoridentification. A phage-displayed peptide library is injected into amouse and allowed to circulate; tissues are harvested and then disruptedinto single cells with bound phage. Non-bound phage are washed away, andbound phage are recovered, grown up in bacteria, and the process iseither repeated or the peptide-encoding inserts are amplified by PCR andshuttled into the bait vector component of the bacterialtwo-hybridization system. Once in the bacteria, the pool enriched forthe ability to home to the selected tissue can interact with possiblereceptors, resulting in carbenicillin-resistance. Resistant clones areplated on X-gal-containing agar for a secondary screen; target and baitinserts are amplified and sequenced from carbenicillin-resistant,LacZ-expressing cells. (B) By the third round of ex vivo selection withanti-CD31 magnetic beads, the selected pool bound ex vivo heart cellsabout 230-fold more than non-recombinant T7 phage. (C) When furtherselected for homing to the heart in vivo, the ex vivo/in vivo selectedpool accumulated in the heart vasculature about 190 times more thannon-recombinant control phage. (D) Peptide-encoding inserts from thefinal pool were subcloned into the bait vector, and bacteria transformedwith both peptide-bait vectors and a heart cDNA library in a targetvector. Transformed bacteria were selected for growth on 500 μg/mlcarbenicillin. (E) Some clones were also positive for the secondarymarker, LacZ, as evidenced by the production of the blue color on platescontaining X-gal.

FIG. 2 shows that individual phage-displayed peptides home selectivelyto the heart. The indicated peptides identified from a combined screenfor heart endothelial homing were reconstituted in T7 phage andindividually administered by intravenous injection into mice. With theexception of SEQ ID NO: 10, phage displaying heart homing peptidessignificantly homed to the heart relative to non-recombinant phage;furthermore, homing was specific for the heart among the major organstested. The highest specificity (over 300-fold) was recorded for theCRPPR (SEQ ID NO: 1)-displaying phage. (A) CRPPR (SEQ ID NO: 1) phagehoming to heart, kidney, brain, muscle and lung. (B) CARPAR (SEQ ID NO:5) phage homing to heart, kidney, brain, muscle and lung. (C) CKRAVR(SEQ ID NO: 7) phage homing to heart, kidney, brain, muscle and lung.(D) CLIDLHVMC (SEQ ID NO: 10) phage homing to heart, kidney, brain,muscle and lung. (E) CRSTRANPC (SEQ ID NO: 11) phage homing to heart,kidney, brain, muscle and lung. (F) CPKTRRVPC (SEQ ID NO: 12) phagehoming to heart, kidney, brain, muscle and lung.

FIG. 3 shows that phage displaying heart-homing peptides co-localizewith a marker of vascular endothelial cells in the heart. Phage wereintravenously injected into mice together with fluorescein-conjugatedtomato lectin (green), and sections of heart tissue stained with rabbitanti-T7 antibody and anti-rabbit-Alexa594 (red). (A) CRPPR (SEQ IDNO: 1) phage staining. (B) CARPAR (SEQ ID NO: 5) phage staining. (C)CKRAVR (SEQ ID NO: 7) phage staining. (D) CLIDLHVMC (SEQ ID NO: 10)phage staining. (E) CRSTRANPC (SEQ ID NO: 11) phage staining. (F)CPKTRRVPC (SEQ ID NO: 12) phage staining. Arrows indicate somerepresentative double-positive blood vessels (yellow). Magnification200×.

FIG. 4 shows that phage displaying heart-homing peptides specificallybind to cells transfected with cognate receptor. Putative receptors forheart homing peptides identified in the two-hybrid screen (Table 1) wereexpressed in 293T cells from transfected cDNA; transfected cells wereused to test binding of cognate phage displaying the relevant hearthoming peptide either in buffer or in the presence of synthetic peptidesrepresenting each of the corresponding peptides (100 mg/ml). Each phagebound to cells expressing its putative cognate receptor (striped column)300 to 500-fold more than to cells transfected with the empty vectorcontrol (gray column). In each case, binding of phage to transfectedcells was inhibited by competition with an excess of cognate peptide(black column), but not by competition with unrelated heart homingpeptides (white columns). Each of the four transfected cell lines wasassayed with free peptide CRPPR (SEQ ID NO: 1), CKRAVR (SEQ ID NO: 7),CRSTRANPC (SEQ ID NO: 1) and CPKTRRVPC (SEQ ID NO: 12). (A) Binding ofphage expressing cognate peptide CRPPR (SEQ ID NO: 1) toCRIP2-transfected cells; (B) Binding of phage expressing cognate peptideCKRAVR (SEQ ID NO: 7) to Sigirr-transfected cells; (C) Binding of phageexpressing cognate peptide CRSTRANPC (SEQ ID NO: 11) to cellstransfected with the MpcII-3-related protein SEQ ID NO: 33; (D) Bindingof phage expressing cognate peptide CPKTRRVPC (SEQ ID NO: 12) to bc10transfected cells.

FIG. 5 shows that the receptors for heart-homing peptides are stronglyexpressed in the heart at the mRNA level and localize to endothelialcells. (A) Real-time PCR results of the four receptor mRNAs in differentmouse heart, lung, spleen, kidney, brain and muscle expressed aspercentage of the level of same mRNA in the heart (mean from twoseparate experiments). (B) Real-time PCR products recovered at the endof the analysis and analyzed by agarose gel electrophoresis. ST=100 bpDNA ladder. (C) Localization of receptors by in situ hybridization(red). HLP/CRIP2, Sigirr/TIR8, MpcII-3-related protein, and bc10 wereall strongly expressed in endothelial cells in the heart. HLP/CRIP2,MpcII-3-related protein, and bc10 mRNAs were also strongly expressed incardiac parenchymal. Lung capillaries were additionally clearly positivefor Sigirr/TIR8 and MpcII-3-related protein, with some signal observedfor HLP/CRIP2 and bc10. Traces of positive staining were occasionallyfound in other tissues. Magnification 200×.

FIG. 6 shows that the CRPPR (SEQ ID NO: 1) peptide and cognate receptorCRIP2 co-localize with CD31 in endothelial cells of heart vessels.Fluorescein-conjugated CRPPR (SEQ ID NO: 1) peptide was intravenouslyinjected into mice, and tissues collected two hours later for stainingwith antibodies against the endothelial marker, CD31. The fluorescentpeptide is shown in green while the CD31 marker is shown in red. (A)Co-localization of SEQ ID NO: 1 and CD31 in heart blood vessels. (B)Co-localization of SEQ ID NO: 1 and CD31 in endocardium. (C) Controlpeptide is not observed in the heart. (D) Anti-CRIP2 antibody stainsheart endothelial cells (red), co-localizing with CD31. (E) Anti-CRIP2antibody co-localizes with injected CRPPR (SEQ ID NO: 1) peptide inheart blood vessels. (F) Anti-CRIP2 antibody co-localizes with injectedCRPPR (SEQ ID NO: 1) peptide in eridocardium.

FIG. 7 shows that CRPPR (SEQ ID NO: 1)-displaying phage bind to heartcells and that the homing of the SEQ ID NO: 1-displaying phage isblocked by anti-CRIP2 antibodies. CRPPR (SEQ ID NO: 1)-displaying phagewere incubated with cell suspensions prepared from the heart; phagebinding was measured in the presence and absence of variousconcentrations of the indicated antibodies or peptides. (A) A chickenanti-CRIP2 antibody blocked SEQ ID NO: 1-displaying phage binding toheart cells; normal IgY was used as the negative control. (B) Dosedependent inhibition of SEQ ID NO: 1-displaying phage binding by freeCRPPR (SEQ ID NO: 1) peptide and lack of inhibition by two unrelatedheart-homing peptides confirmed the specificity of the ex vivo phagebinding system. (C) In vivo homing of the CRPPR (SEQ ID NO:1)-displaying phage to the heart was inhibited by co-injected anti-CRIP2antibody. Shown are the mean and standard deviation from three separateexperiments.

FIG. 8 shows (A) the nucleotide (SEQ ID NO: 24) and (B) the amino acid(SEQ ID NO: 25) sequence of murine cysteine-rich protein 2 (HLP/CRIP2).Genbank accession NM_(—)024223.

FIG. 9 shows (A) the nucleotide (SEQ ID NO: 26) and (B) the amino acid(SEQ ID NO: 27) sequence of the murine RIKEN EST identified as receptorclone 9. Genbank accession BC026536.

FIG. 10 shows (A) the nucleotide (SEQ ID NO: 28) and (B) the amino acid(SEQ ID NO: 29) sequence of murine single Ig IL-2 receptor relatedprotein (Sigirr/TIR8). Genbank accession BC010806.

FIG. 11 shows the nucleotide sequence (SEQ ID NO: 30) of the murineolfactory cDNA which is a glutamine-rich region containing proteinidentified as receptor clone 27. Genbank accession AK032239.

FIG. 12 shows (A) the nucleotide (SEQ ID NO: 32) and (B) the amino acid(SEQ ID NO: 33) sequence of the murine polypeptide having putativesimilarity to integral membrane protein CII-3 (MpcII-3). Genbankaccession AK032458.

FIG. 13 shows (A) the nucleotide (SEQ ID NO: 34) and (B) the amino acid(SEQ ID NO: 35) sequence of murine bladder cancer-associated proteinhomolog (bc10). Genbank accession BC026935.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification of molecules whichhome to heart vasculature with high selectivity and which can be usefulin the form of conjugates for selectively targeting a systemicallyadministered therapeutic or imaging agent to heart vasculature. Suchselective targeting of a therapeutic agent increases the effectiveamount of the agent delivered to heart vasculature while reducing thelikelihood that the agent will have an adverse effect on other organs.The present invention further relates to identification of cognatereceptors for the homing molecules of the invention. As disclosedfurther below, these receptors are expressed on heart endothelium andcan be useful for screening for optimizing the characteristics ofpeptides or other molecules that selectively home to heart vasculature.

As disclosed herein in Example I, a novel combination of ex vivo and invivo phage selection and bacterial two-hybridization analysis was usedto identify peptides that selectively home to heart vasculature, and toidentify the endothelial molecules which serve as receptors for thesepeptides. Following ex vivo phage selection with murine heart cellsuspensions and anti-CD31 magnetic beads, in vivo selection for homingto the heart was performed, resulting in a phage pool that homed to theheart with an increase of nearly 200-fold relative to non-recombinantphage (FIGS. 1A and 1B). Furthermore, as shown in FIG. 1C, the exvivo/in vivo selected phage preferentially localized to the heart ascompared to other tissues; the enrichment in heart was 20 to 50-foldgreater than the accumulation in brain, kidney, skin and skeletalmuscle.

Of 25 putative receptor clones identified by bacterial two-hybridanalysis with the pool of heart-homing phage, the majority were proteinsabundantly expressed in cardiac muscle. As summarized in Table 1, theremaining six clones represented membrane or cell surface proteins andwere putative receptors for the homing peptides. A first clone, denotedreceptor clone 5, represents the carboxy-terminal 92 amino acids(residues 117 to 208) of heart LIM-protein (HLP), also designatedcysteine-rich protein 2 (CRIP2). The full length murine HLP/CRIP2nucleic acid and amino acid sequences are provided herein as SEQ ID NOS:24 and 25, respectively (see Genbank accession NM_(—)024223). Two-hybridanalysis yielded several potential heart-homing peptides which boundthis receptor. When assayed individually, CRPPR (SEQ ID NO:1)-displaying phage homed to the heart with more than 300 foldselectivity relative to non-recombinant phage (FIG. 2A), and CGRKSKTVC(SEQ ID NO: 2)-phage displayed about 50-fold selectivity in heart-homingrelative to non-recombinant phage. These results indicate that peptidesSEQ ID NO: 1 and SEQ ID NO: 2 are heart-homing peptides and furthersuggest that HLP/CRIP2 serves as the receptor for homing of thesepeptides.

As further disclosed herein, an unannotated RIKEN EST (receptor clone9), was also identified as a membrane or cell surface-expressed proteinwhich binds to one or more heart-homing peptides. The full-length murinenucleic acid and amino acid sequences of the RIKEN EST represented byreceptor clone 9 are provided herein as SEQ ID NOS: 26 and 27,respectively (see Genbank accession BC026536). Of three distinctpeptides identified through two-hybrid analysis, two peptides, CARPAR(SEQ ID NO: 5) and CPKRPR (SEQ ID NO: 6), exhibited selective homing tothe heart when displayed on phage (FIG. 2B).

Results disclosed herein further identified the single Ig IL-1 receptorrelated protein designated Sigirr or TIR8 (receptor clone 15), as areceptor for heart homing peptides. The full length murine Sigirr/TIR8nucleic acid and amino acid sequences are provided herein as SEQ ID NOS:28 and 29, respectively (see Genbank accession BC010806). As summarizedin Table 1, three peptides identified by binding Sigirr/TIR8 throughtwo-hybrid analysis, CKRAVR (SEQ ID NO: 7), CRNSWKPNC (SEQ ID NO: 8),and RGSSS (SEQ ID NO: 9), showed 20 to 30-fold heart homing selectivitywhen displayed on phage and assayed in vivo (see Table 1 and FIG. 2C).

Also disclosed herein as a heart-homing receptor is receptor clone 36,which represents an unnamed protein product from the RIKEN Fantom setwith putative similarity to integral membrane protein CII-3 (MpcII-3), amitochondrial membrane protein which is part of the succinatedehydrogenase complex. The nucleic acid and amino acid sequences of thisMpcII-3-related protein are provided herein as SEQ ID NOS: 32 and 33,respectively (see Genbank accession 26328274). Through two-hybridanalysis, the peptide CRSTRANPC (SEQ ID NO: 11) was identified asbinding to this receptor; phage displaying SEQ ID NO: 11 homed to theheart with about 20-fold selectivity when assayed individually in vivo(Table I and FIG. 2E). Additional results disclosed herein indicate thatmurine bladder cancer-associated protein homolog 10 (bc10), a smallmembrane protein which is down regulated as cancer develops frompre-malignant lesions in the bladder, can serve as a heart homingreceptor. The identified receptor clone 46 represents a portion of themouse homolog of bc10. The full length murine bc10 nucleic acid andamino acid sequences are provided herein as SEQ ID NOS: 34 and 35,respectively (see Genbank accession 20072483). As shown in Table 1 andFIG. 2F, phage expressing two peptides that bound clone 46, CPKTRRVPC(SEQ ID NO: 12) and CSGMARTKC (SEQ ID NO: 13), demonstrated about 60-and 10-fold selectivity in heart homing, respectively.

As additionally disclosed herein, several heart-homing peptides wereanalyzed for the ability to bind heart endothelia in vivo and forspecific binding to their putative receptors. Phage-displaying the hearthoming peptides SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11and SEQ ID NO: 12 were individually injected into mice withfluorescein-conjugated tomato lectin, a blood vessel marker, andlocalization of the phage observed with anti-T7 antibody. As shown inFIGS. 3A to C, E and F, phage displaying SEQ ID NOS: 1, 5, 7, 11 or 12were present in heart endothelia and absent from other tissues, while anegative control phage, which displayed peptide SEQ ID NO: 10, wasabsent from the heart and other tissues. These results demonstrate thatphage displaying peptides CRPPR (SEQ ID NO: 1), CARPAR (SEQ ID NO: 5),CKRAVR (SEQ ID NO: 7), CRSTRANPC (SEQ ID NO: 11) or CPKTRRVPC (SEQ IDNO: 12) localize to heart vasculature in vivo. In addition, real time(RT)-PCR and in situ hybridization analysis showed that four cognatereceptors for heart homing peptides, HLP/CRIP2, Sigirr/TIR8,MpcII-3-related protein and bc10, were expressed in the heartendothelia. These receptors were also preferentially expressed in theheart as compared to other tissues assayed (see FIG. 5). Although theheart endothelial receptors HLP/CRIP2, Sigirr/TIR8, MpcII-3-relatedprotein and bc10 were not expressed exclusively in the heart, theirpreferential expression in the heart resulted in homing peptides whichconcentrated test phage in the heart with a selectivity of about 20- to300-fold. This 20- to 300-fold selectivity in heart homing issignificantly increased as compared to the heart homing previouslyobserved with other molecules.

As further disclosed herein, phage displaying cognate peptides wereassayed for the ability to bind to cells transfected with full-lengthcognate receptor. As shown in FIG. 4, phage expressing the heart homingpeptides bound to cells transfected with the corresponding cognatereceptor 300 to 500-fold more than control phage, and phage binding wasinhibited in the presence of 100 μg/ml cognate peptide but not byunrelated homing peptide. These results confirm that the heart homingpeptide CRPPR (SEQ ID NO: 1) specifically binds the receptor HLP/CRIP2;the heart homing peptide CKRAVR (SEQ ID NO: 7) specifically binds thereceptor Sigirr/TIR8; the heart homing peptide CRSTRANPC (SEQ ID NO: 11)specifically binds the MpcII-3-related protein receptor; and the hearthoming peptide CPKTRRVPC (SEQ ID NO: 12) specifically binds the receptorbc10.

Additional results disclosed herein demonstrate co-localization ofpeptide CRPPR (SEQ ID NO: 1) with cognate receptor HLP/CRIP2. As shownherein in FIG. 6, fluorescence from intravenously injected CRPPR (SEQ IDNO: 1) peptide co-localized extensively with the vascular marker CD31 inboth heart blood vessels and endocardium, while a fluorescein-labeledcontrol peptide was not detected in the heart. Anti-HLP/CRIP2 antibodystaining also co-localized with the vascular marker (FIG. 6D) andpeptide CRPPR (SEQ ID NO: 1; FIGS. 6E and 6F) in heart vessels and heartendocardium. The specificity of CRPPR (SEQ ID NO: 1)-phage binding toheart vessels with HLP/CRIP2 was demonstrated using competition assayswith free CRPPR (SEQ ID NO: 1) peptide, which blocked binding of CRPPR(SEQ ID NO: 1)-displaying phage to heart-derived cell suspensions in adose-dependent manner (FIGS. 7A and B). In addition, anti-HLP/CRIP2antibody inhibited CRPPR (SEQ ID NO: 1)-phage homing when co-injectedwith the phage (FIG. 7C). These results substantiate that peptide CRPPR(SEQ ID NO: 1) selectively homes to heart vasculature through specificbinding to HLP/CRIP2.

Based on the above findings, the present invention provides a conjugatecontaining a therapeutic agent linked to a homing molecule thatselectively homes to heart vasculature and specifically bindscysteine-rich protein 2 (HLP/CRIP2; SEQ ID NO: 25). In such a conjugate,the homing molecule can home to the heart in vivo with a selectivity,for example, of at least 5-fold relative to non-recombinant phage, andcan be, for example, a peptide or peptidomimetic. In one embodiment, aconjugate of the invention includes a homing peptide or peptidomimeticcontaining the amino acid sequence CRPPR (SEQ ID NO: 1) or aconservative variant or peptidomimetic thereof. In another embodiment, aconjugate of the invention includes a homing peptide or peptidomimeticthat contains the amino acid sequence CGRKSKTVC (SEQ ID NO: 2) or aconservative variant or peptidomimetic thereof. Such a homing peptide orpeptidomimetic optionally can be conformationally constrained.

A variety of therapeutic agents are useful in the conjugates of theinvention which incorporate a homing molecule that selectively homes toheart vasculature and specifically binds HLP/CRIP2 (SEQ ID NO: 25).Useful therapeutic agents include, without limitation, angiogenicagents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzyme (ACE)inhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors. In one embodiment, the invention provides a conjugatewhich contains an angiogenic agent linked to a homing molecule thatselectively homes to heart vasculature and specifically binds HLP/CRIP2(SEQ ID NO: 25). In another embodiment, the invention provides aconjugate which contains an anti-thrombotic agent linked to a homingmolecule that selectively homes to heart vasculature and specificallybinds HLP/CRIP2 (SEQ ID NO: 25).

The present invention additionally provides a conjugate containing atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature and specifically binds receptor clone 9 (SEQ ID NO:27). In one embodiment, a conjugate of the invention includes a homingpeptide or peptidomimetic containing the amino acid sequence CARPAR (SEQID NO: 5) or a conservative variant or peptidomimetic thereof. Inanother embodiment, a conjugate of the invention includes a homingpeptide or peptidomimetic that contains the amino acid sequence CPKRPR(SEQ ID NO: 6) or a conservative variant or peptidomimetic thereof. Anyof a variety of therapeutic agents are useful in the conjugates of theinvention which incorporate a homing molecule that selectively homes toheart vasculature and specifically binds receptor clone 9 (SEQ ID NO:27). Useful therapeutic agents include, without limitation, angiogenicagents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors. In one embodiment, the invention provides a conjugatewhich contains an angiogenic agent linked to a homing molecule thatselectively homes to heart vasculature and specifically binds receptorclone 9 (SEQ ID NO: 27). In another embodiment, the invention provides aconjugate which contains an anti-thrombotic agent linked to a homingmolecule that selectively homes to heart vasculature and specificallybinds receptor clone 9 (SEQ ID NO: 27).

Further provided herein is a conjugate containing a therapeutic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds single Ig IL-2 receptor related protein(Sigirr/TIR8; SEQ ID NO: 29). As a non-limiting example, a homingmolecule useful in such a conjugate can home to the heart in vivo with aselectivity of at least 5-fold relative to non-recombinant phage. As afurther non-limiting example, a homing molecule that specifically bindsSigirr/TIR8 can be a homing peptide or peptidomimetic.

A variety of homing peptides and peptidomimetics that specifically bindSigirr/TIR8 (SEQ ID NO: 29) are useful in the conjugates of theinvention, including, without limitation, homing peptides orpeptidomimetics containing the amino acid sequence CKRAVR (SEQ ID NO: 7)or a conservative variant or peptidomimetic thereof. Homing peptides andpeptidomimetics useful in the invention further include, withoutlimitation, peptides and peptidomimetics containing the amino acidsequence CRNSWKPNC (SEQ ID NO: 8) or a conservative variant orpeptidomimetic thereof; such peptides and peptidomimetics may or may notbe conformationally constrained. Additional homing peptides andpeptidomimetics useful in the invention include, without limitation,those containing the amino acid sequence RGSSS (SEQ ID NO: 9) or aconservative variant or peptidomimetic thereof.

Any of a variety of therapeutic agents are useful in conjugates thatcontain a therapeutic agent linked to a homing molecule that selectivelyhomes to heart vasculature and specifically binds Sigirr/TIR8 (SEQ IDNO: 29). Therapeutic agents useful in the invention include, yet are notlimited to, angiogenic agents, anti-thrombotic agents, anti-inflammatoryagents, immunosuppressive agents, anti-arrhythmic agents, tumor necrosisfactor inhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors. In one embodiment, the invention provides a conjugatewhich contains an angiogenic agent linked to a homing molecule thatselectively homes to heart vasculature and specifically bindsSigirr/TIR8 (SEQ ID NO: 29). In another embodiment, the inventionprovides a conjugate which contains an anti-thrombotic agent linked to ahoming molecule that selectively homes to heart vasculature andspecifically binds Sigirr/TIR8 (SEQ ID NO: 29).

Also provided herein is a conjugate which contains a therapeutic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds SEQ ID NO: 33. In one embodiment, a conjugate ofthe invention includes a homing molecule that homes to the heart in vivowith a selectivity of at least 5-fold relative to non-recombinant phage.In another embodiment, a conjugate of the invention includes a homingmolecule which is a homing peptide or peptidomimetic. In furtherembodiments, a conjugate of the invention includes a homing peptide orpeptidomimetic that contains the amino acid sequence CRSTRANPC (SEQ IDNO: 11) or a conservative variant or peptidomimetic thereof. Such ahoming molecule can optionally be conformationally constrained. It isunderstood that any of a variety of therapeutic agents can be useful inthe conjugates of the invention including, without limitation,angiogenic agents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors. As one example, a conjugate of the invention can containan angiogenic agent linked to a homing molecule that selectively homesto heart vasculature and specifically binds SEQ ID NO: 33. As anotherexample, a conjugate of the invention can contain an anti-thromboticagent linked to a homing molecule that selectively homes to heartvasculature and specifically binds SEQ ID NO: 33.

Also provided herein is a conjugate containing a therapeutic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds murine bladder cancer-associated protein homolog(bc10; SEQ ID NO: 35). In such a conjugate, the homing molecule can hometo the heart in vivo with a selectivity, for example, of at least 5-foldrelative to non-recombinant phage. The homing molecule can be, withoutlimitation, a peptide or peptidomimetic. Any of a variety of homingmolecules that specifically bind bc10 (SEQ ID NO: 35) can be useful inthe conjugates of the invention including, without limitation, homingpeptides and peptidomimetics that contain the amino acid sequenceCPKTRRVPC (SEQ ID NO: 12) or a conservative variant or peptidomimeticthereof. Useful homing molecules that specifically bind bc10 (SEQ ID NO:35) further include, without limitation, homing peptides andpeptidomimetics that contain the amino acid sequence CSGMARTKC (SEQ IDNO: 13) or a conservative variant or peptidomimetic thereof. Usefulhoming molecules further include, without limitation, conformationallyconstrained forms of homing peptides and peptidomimetics including theamino acid sequence SEQ ID NO: 12 or SEQ ID NO: 13 or a conservativevariant or peptidomimetic thereof.

One skilled in the art understands that any of a variety of therapeuticagents can be useful in the conjugates of the invention including,without limitation, angiogenic agents, anti-thrombotic agents,anti-inflammatory agents, immunosuppressive agents, anti-arrhythmicagents, tumor necrosis factor inhibitors, endothelin inhibitors,angiotensin-converting enzyme inhibitors, calcium antagonists,antibiotic agents, antiviral agents and viral vectors. In oneembodiment, a conjugate of the invention contains an angiogenic agentlinked to a homing molecule that selectively homes to heart vasculatureand specifically binds bc10 (SEQ ID NO: 35). In another embodiment, aconjugate of the invention contains an anti-thrombotic agent linked to ahoming molecule that selectively homes to heart vasculature andspecifically binds bc10 (SEQ ID NO: 35).

In particular embodiments, the peptide or peptidomimetic portion of aconjugate of the invention has a defined length. The peptide orpeptidomimetic portion of the conjugate can have, without limitation, alength of at most 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1000 or 2000 residues. In other embodiments, thepeptide or peptidomimetic portion of the conjugate has a length of atleast 5, 10, 20, 30, 50, 100, 150, 200, 250, or 300 residues. It isunderstood that the term “peptide or peptidomimetic portion of theconjugate” means the total number of residues in the homing peptide orpeptidomimetic and any contiguous protein, peptide or peptidomimetic,such as a therapeutic protein or pro-apoptotic peptide.

As disclosed herein, peptides SEQ ID NOS: 1 and 2 recognize a target“receptor” which is expressed on heart endothelium and which is notexpressed to the same extent in other tissues. The endothelial andtissue selective expression of this target receptor, identified hereinas HLP/CRIP2, form the basis for the selective homing activity ofpeptides SEQ ID NOS: 1 and 2 and related peptides and peptidomimetics,as well as other molecules with similar binding specificity. Based onthese discoveries, it is clear that molecules structurally unrelated toSEQ ID NOS: 1 and 2 but with specific HLP/CRIP2 binding activity alsowill share the characteristic of selectively homing to heartvasculature. Such molecules can be identified, for example, by theability to specifically bind to, or to compete with SEQ ID NO: 1 or 2for binding to, HLP/CRIP2-expressing cells such as the 293T cellstransfected with HLP/CRIP2 disclosed herein. Selective homing to heartvasculature can be readily confirmed, for example, as described hereinin Example II.

Thus, the present invention provides a homing molecule that selectivelyhomes to heart vasculature and specifically binds cysteine-rich protein2 (HLP/CRIP2; SEQ ID NO: 25). In one embodiment, the homing moleculewhich specifically binds HLP/CRIP2 (SEQ ID NO: 25) is a molecule otherthan an antibody or antigen-binding fragment thereof. In a furtherembodiment, the homing molecule which specifically binds HLP/CRIP2 (SEQID NO: 25) is a peptide or peptidomimetic. In addition to the HLP/CRIP2(SEQ ID NO: 25)-binding molecules disclosed herein, CRPPR (SEQ ID NO: 1)and CGRKSKTVC (SEQ ID NO: 2), screening methods for identification ofadditional, structurally related or unrelated, homing molecules also areprovided herein below.

Additional target receptors for molecules that selectively home to heartvasculature also have been identified herein. Receptor clone 9 (SEQ IDNO: 27) has been identified as a receptor which specifically binds theheart homing peptides SEQ ID NOS: 5 and 6, and forms the basis for theability of these peptides and additional peptides, peptidomimetics andother molecules with similar binding activity to selectively home toheart vasculature. From the above, it is clear that moleculesstructurally unrelated to SEQ ID NOS: 5 or 6 but with specific receptorclone 9 (SEQ ID NO: 27)-binding activity also share the characteristicof selectively homing to heart vasculature. Such homing molecules can beidentified by the ability to specifically bind to, or to compete withSEQ ID NO: 5 or 6 for binding to, receptor clone 9 (SEQ ID NO: 27).Specific binding can be assayed using purified SEQ ID NO: 27 or, forexample, cells transfected with a SEQ ID NO: 27-encoding nucleic acidmolecule such as SEQ ID NO: 26.

Based on the above, the invention provides a homing molecule thatselectively homes to heart vasculature and specifically binds receptorclone 9 (SEQ ID NO: 27). In one embodiment, the homing molecule whichspecifically binds receptor clone 9 (SEQ ID NO: 27) is a molecule otherthan an antibody or antigen-binding fragment thereof. In a furtherembodiment, the homing molecule which specifically binds receptor clone9 (SEQ ID NO: 27) is a peptide or peptidomimetic. Exemplary peptides andpeptidomimetics which specifically bind receptor clone 9 (SEQ ID NO: 27)are disclosed herein, and screening methods suitable for identificationof additional homing molecules that selectively home to heartvasculature and specifically bind receptor clone 9 (SEQ ID NO: 27) areprovided below.

As further disclosed herein, the endothelial and heart-selectiveexpression of Sigirr/TIR8, a polypeptide which specifically binds SEQ IDNOS: 7, 8 and 9, forms the basis for the ability of these peptides andadditional peptides, peptidomimetics and other molecules with similarbinding activity to selectively home to heart vasculature. From theabove, it is clear that molecules structurally unrelated to SEQ ID NOS:7, 8 or 9 but with specific Sigirr/TIR8 binding activity also share thecharacteristic of selectively homing to heart vasculature. Such homingmolecules can be identified by the ability to specifically bind to, orto compete with SEQ ID NO: 7, 8 or 9 for binding to, Sigirr/TIR8.Specific binding can be assayed using purified Sigirr/TIR8 or, forexample, cells such as Sigirr/TIR8-transfected 293T cells disclosed inthe examples below.

Based on identification of Sigirr/TIR8 as a heart-homing receptor, thepresent invention provides a homing molecule that selectively homes toheart vasculature and specifically binds Sigirr/TIR8 (SEQ ID NO: 29). Inone embodiment, the homing molecule which specifically binds Sigirr/TIR8(SEQ ID NO: 29) is a molecule other than an antibody or antigen-bindingfragment thereof. In a further embodiment, the homing molecule whichspecifically binds Sigirr/TIR8 (SEQ ID NO: 29) is a peptide orpeptidomimetic. Exemplary peptides and peptidomimetics whichspecifically bind Sigirr/TIR8 (SEQ ID NO: 29) are disclosed herein.Furthermore, screening methods suitable for routine identification ofadditional homing molecules that selectively home to heart vasculatureand specifically bind Sigirr/TIR8 (SEQ ID NO: 29) are provided hereinbelow.

Peptide CRSTRANPC (SEQ ID NO: 11) recognizes another target “receptor”which is selectively expressed on heart endothelium. Due to theendothelial and tissue selective expression of the MpcII-3-relatedprotein SEQ ID NO: 33, peptide SEQ ID NO: 11 as well as relatedpeptides, peptidomimetics and other molecules with similar bindingspecificity can selectively home to heart vasculature. Such homingmolecules include those which are structurally unrelated to peptide SEQID NO: 11 but which also specifically bind to the MpcII-3 relatedprotein SEQ ID NO: 33. In view of the above, it is clear that additionalmolecules which selectively home to heart vasculature can be identifiedby their ability to specifically bind to, or to compete with SEQ ID NO:11 for binding to, a MpcII-3-related protein, including purifiedMpcII-3-related protein or cells expressing a MpcII-3-related protein onthe cell surface.

From the finding that MpcII-3-related protein (SEQ ID NO: 33) is aheart-homing receptor, the present invention provides a homing moleculethat selectively homes to heart vasculature and specifically binds SEQID NO: 33. In one embodiment, the homing molecule which specificallybinds SEQ ID NO: 33 is a molecule other than an antibody orantigen-binding fragment thereof. In another embodiment, the homingmolecule which specifically binds SEQ ID NO: 33 is a peptide orpeptidomimetic. Exemplary peptides and peptidomimetics whichspecifically bind SEQ ID NO: 33 are disclosed herein, as are screeningmethods suitable for identification of additional homing molecules thatselectively home to heart vasculature and specifically bind SEQ ID NO:33.

Another target receptor, bc10, was further identified herein through thespecific binding activity of SEQ ID NOS: 12 and 13. The endothelial andheart-selective expression of this receptor form the basis for theselective homing activity of peptides SEQ ID NOS: 12 and 13 as well asrelated peptides, peptidomimetics and other molecules with similarbinding specificity. Based on the findings disclosed herein, one skilledin the art understands that molecules structurally unrelated to SEQ IDNOS: 12 and 13 also can selectively home to heart vasculature when theyshare the characteristic of specifically binding bc10. Thus, additionalhoming molecules can be identified, for example, by the ability tospecifically bind to, or to compete with SEQ ID NO: 12 or 13 for bindingto, target receptor bc10, which can be provided, for example, in theform of purified bc10 or bc10-expressing cells such as bc10-transfected293T cells. Selective homing to heart vasculature can be readilyconfirmed, for example, as described herein in Example II.

Based on the discovery that bc10 is the cognate receptor for peptidesSEQ ID NOS: 12 and 13, the present invention provides a homing moleculethat selectively homes to heart vasculature and specifically binds bc10(SEQ ID NO: 35). In one embodiment, the invention provides a homingmolecule which specifically binds bc10 (SEQ ID NO: 35) and which is amolecule other than an antibody or antigen-binding fragment thereof. Inanother embodiment, the invention provides a homing molecule whichspecifically binds bc10 (SEQ ID NO: 35) and which is a peptide orpeptidomimetic. Peptides SEQ ID NOS: 12 and 13 and conservative variantsthereof are disclosed herein as molecules which specifically bind bc10(SEQ ID NO: 35). In addition, the screening methods disclosed furtherbelow are suitable for identification of other homing molecules,including but not limited to, homing peptides and peptidomimetics.

For convenience, the five target receptors identified herein, HLP/CRIP2,receptor clone 9, Sigirr/TIR8, MpcII-3-related protein, and bc10, arecollectively termed “heart-homing receptors.”

As indicated above, the present invention provides a variety of homingmolecules that selectively home to heart vasculature. As used herein,the term “molecule” is used broadly to mean a polymeric or non-polymericorganic chemical such as a small molecule drug; a nucleic acid moleculesuch as an RNA, a cDNA or an oligonucleotide; a peptide orpeptidomimetic; or a protein such as an antibody or a growth factorreceptor or a fragment thereof such as an Fv, Fd, or Fab fragment of anantibody containing the antigen-binding domain. In one embodiment, themolecule is an organic chemical other than an antibody orantigen-binding fragment thereof. In another embodiment, the molecule isa peptide or peptidomimetic.

The term “homing molecule” as used herein, means any molecule thatselectively localizes in vivo to heart vasculature in preference to mostother tissues and vasculature. Similarly, the term “homing peptide” or“homing peptidomimetic” means a peptide or peptidomimetic thatselectively localizes in vivo to the heart vasculature in preference tomost other tissues and vasculature.

The term “selectively homes,” as used herein in reference to a molecule,means that, in vivo, the homing molecule localizes preferentially toheart vasculature as compared to most other tissues or vasculature.Selective homing generally is characterized by at least a two-foldgreater localization in heart vasculature as compared to other tissuessuch as brain, lung, kidney and muscle. A homing molecule can becharacterized by 5-fold, 10-fold, 20-fold or more preferentiallocalization to heart vasculature as compared to many or most non-tumortissues. It is understood that a homing molecule can home, in part, tovasculature or tissue outside the heart or to a small population ofcells outside of the heart in addition to selectively homing to heartvasculature.

As discussed above, a homing molecule of the invention specificallybinds one of the heart-homing receptors of the invention. As usedherein, the term “specifically binds” or “specifically binding” meansbinding that is measurably different from a non-specific interaction.Specific binding can be measured, for example, by determining binding ofa molecule compared to binding of a control molecule, which generally isa molecule of similar structure that does not have binding activity.Specific binding also can be indicated if the homing molecule hasmeasurably higher affinity for cells transfected with cognateheart-homing receptor (HLP/CRIP2, receptor clone 9, Sigirr/TIR8,MpcII-3-related protein or bc10) than for cells that do not express thecognate receptor. The measurably higher affinity can be, for example, anincrease of at least 100-fold, 200-fold, 300-fold, 500-fold or more forcells transfected with cognate receptor as compared to control cells.Binding specificity also can be confirmed, for example, by competitiveinhibition with a known receptor-binding molecule.

Furthermore, the term specifically binding, as used herein, encompassesboth low and high affinity specific binding. Specific binding can beexhibited, for example, by a low affinity homing molecule having a Kd ofat least about 10⁻⁴ M. For example, if a heart-homing receptor has morethan one binding site, a homing molecule having low affinity can beuseful for targeting heart vasculature. Specific binding also can beexhibited by a high affinity homing molecule, for example, a homingmolecule having a Kd of at least about 10⁻⁵ M. Such a molecule can have,for example, a Kd of at least about 10⁻⁶ M, at least about 10⁻⁷ M, atleast about 10⁻⁸ M, at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, orcan have a Kd of at least about 10⁻¹¹ M or 10⁻¹² M or greater. Both lowand high affinity homing molecules are useful and encompassed by theinvention. Low affinity homing molecules can be useful in targeting,without limitation, multivalent conjugates including viruses and otherparticles. High affinity homing molecules are useful in targeting,without limitation, multivalent and univalent conjugates.

Also provided herein are multivalent conjugates, which incorporate atleast two homing molecules that each selectively homes to heartvasculature. In particular embodiments, a multivalent conjugate of theinvention includes at least ten or at least 100 of such homingmolecules. A variety of therapeutic agents are useful in the multivalentconjugates of the invention including, but not limited to, phage andother therapeutic agents described further below. In one embodiment, theinvention provides a multivalent conjugate containing at least twohoming peptides or peptidomimetics that each selectively homes to heartvasculature and specifically binds HLP/CRIP2 (SEQ ID NO: 25), receptorclone 9 (SEQ ID NO: 27), Sigirr/TIR8 (SEQ ID NO: 29), MpcII-3-relatedprotein (SEQ ID NO: 33) or bc10 (SEQ ID NO: 35). In another embodiment,such a conjugate contains at least ten homing peptides orpeptidomimetics that each selectively homes to heart vasculature andspecifically binds HLP/CRIP2 (SEQ ID NO: 25), receptor clone 9 (SEQ IDNO: 27), Sigirr/TIR8 (SEQ ID NO: 29), MpcII-3-related protein (SEQ IDNO: 33) or bc10 (SEQ ID NO: 35). In still another embodiment, aconjugate of the invention contains at least 100 homing peptides orpeptidomimetics that each selectively homes to heart vasculature andspecifically binds HLP/CRIP2 (SEQ ID NO: 25), receptor clone 9 (SEQ IDNO: 27), Sigirr/TIR8 (SEQ ID NO: 29), MpcII-3-related protein (SEQ IDNO: 33) or bc10 (SEQ ID NO: 35).

In specific embodiments, a multivalent conjugate of the inventionincludes two or more, three or more, five or more, ten or more, twentyor more, thirty or more, forty or more, fifty or more, 100 or more, 200or more, 300 or more, 400 or more, 500 or more or 100 or more homingmolecules that selectively home to heart vasculature and specificallybind HLP/CRIP2 (SEQ ID NO: 25), receptor clone 9 (SEQ ID NO: 27),Sigirr/TIR8 (SEQ ID NO: 29), MpcII-3-related protein (SEQ ID NO: 33) orbc10 (SEQ ID NO: 35). In one embodiment, the homing moleculesspecifically bind the same heart-homing receptor. In another embodiment,the homing molecules have an identical amino acid sequence. In a furtherembodiment, the multivalent conjugate includes homing molecules havingnon-identical amino acid sequences. Moieties useful in a multivalentconjugate of the invention that incorporates multiple homing moleculesinclude, but are not limited to, phage; retroviruses; adenoviruses;adeno-associated viruses and other viruses; cells; liposomes; polymericmatrices; non-polymeric matrices or particles such as gold particles;microdevices; nanodevices; and nano-scale semiconductor materials.

A multivalent conjugate of the invention can contain, for example, aliposome or other polymeric matrix linked to at least two homingmolecules that each selectively homes to heart vasculature andspecifically binds HLP/CRIP2 (SEQ ID NO: 25), receptor clone 9 (SEQ IDNO: 27), Sigirr/TIR8 (SEQ ID NO: 29), MpcII-3-related protein (SEQ IDNO: 33) or bc10 (SEQ ID NO: 35). If desired, the liposome or otherpolymeric matrix can be linked to at least ten or at least 100 of suchhoming molecules. Homing molecules useful in such a multivalentconjugate can independently include, for example, the amino acidsequence SEQ ID NO: 1, 2, 7, 8, 9, 11, 12 or 13 or a conservativevariant or peptidomimetic of such a sequence. Liposomes composed, forexample, of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press,Boca Raton, Fla. (1984)). One skilled in the art understands that, in amultivalent conjugate of the invention, the liposome or other polymericmatrix additionally can include another component if desired such as,without limitation, a therapeutic agent, anti-angiogenic agent,anti-inflammatory agent or immunosuppressive agent.

The present invention provides a method of treating a cardiovasculardisease in a subject by administering to the subject an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and that specifically binds HLP/CRIP2 (SEQ ID NO: 25). Theinvention further provides a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to an antibody, or antigen-bindingfragment thereof, that selectively homes to heart vasculature and thatspecifically binds HLP/CRIP2 (SEQ ID NO: 25), thereby directing themoiety to heart vasculature. Antibodies useful in the methods of theinvention include, without limitation, monoclonal antibodies andantibodies with biological activity. Where a method of the invention isused to direct a moiety to heart vasculature, the moiety can be, withoutlimitation, a detectable moiety or a therapeutic agent such as, withoutlimitation, an angiogenic agent, anti-thrombotic agent,anti-inflammatory agent, immunosuppressive agent, anti-arrhythmic agent,tumor necrosis factor inhibitor, endothelin inhibitor,angiotensin-converting enzyme inhibitor, calcium antagonist, antibioticagent, antiviral agent or viral vector. Moieties and cardiovasculardiseases are described further herein below.

The present invention also provides a method of treating acardiovascular disease in a subject by administering to the subject anantibody, or antigen-binding fragment thereof, that selectively homes toheart vasculature and that specifically binds SEQ ID NO: 27. Theinvention additionally provides a method of directing a moiety to heartvasculature in a subject in which a conjugate is administered to asubject, the conjugate containing a moiety linked to an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and specifically binds SEQ ID NO:27, thereby directing themoiety to heart vasculature. In such methods of the invention, theantibody can be, without limitation, a monoclonal antibody or anantibody with biological activity. Where a method of the invention isused to direct a moiety to heart vasculature, the moiety can be, withoutlimitation, a detectable moiety or a therapeutic agent such as, withoutlimitation, an angiogenic agent, anti-thrombotic agent,anti-inflammatory agent, immunosuppressive agent, anti-arrhythmic agent,tumor necrosis factor inhibitor, endothelin inhibitor,angiotensin-converting enzyme inhibitor, calcium antagonist, antibioticagent, antiviral agent or viral vector.

The present invention also provides a method of treating acardiovascular disease in a subject by administering to the subject anantibody, or antigen-binding fragment thereof, that selectively homes toheart vasculature and that specifically binds Sigirr/TIR8 (SEQ ID NO:29). In addition, there is provided herein a method of directing amoiety to heart vasculature in a subject by administering to the subjecta conjugate which contains a moiety linked to an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and that specifically binds Sigirr/TIR8 (SEQ ID NO: 29),thereby directing the moiety to heart vasculature. In the methods of theinvention, the antibody can be, without limitation, a monoclonalantibody or an antibody with biological activity. As discussed above,where a method of the invention is used to direct a moiety to heartvasculature, the moiety can be, without limitation, a detectable moietyor a therapeutic agent such as, without limitation, an angiogenic agent,anti-thrombotic agent, anti-inflammatory agent, immunosuppressive agent,anti-arrhythmic agent, tumor necrosis factor inhibitor, endothelininhibitor, angiotensin-converting enzyme inhibitor, calcium antagonist,antibiotic agent, antiviral agent or viral vector.

Further provided herein is a method of treating a cardiovascular diseasein a subject by administering to the subject an antibody, orantigen-binding fragment thereof, that selectively homes to heartvasculature and that specifically binds SEQ ID NO: 33. Also provided bythe invention is a method of directing a moiety to heart vasculature ina subject by administering to the subject a conjugate which contains amoiety linked to an antibody, or antigen-binding fragment thereof, thatselectively homes to heart vasculature and that specifically binds SEQID NO: 33, thereby directing the moiety to heart vasculature. Antibodiesuseful in the invention include, without limitation, monoclonalantibodies as well as antibodies with biological activity. In themethods of the invention for directing a moiety to heart vasculature,the moiety can be, without limitation, a detectable moiety or atherapeutic agent such as, without limitation, an angiogenic agent,anti-thrombotic agent, anti-inflammatory agent, immunosuppressive agent,anti-arrhythmic agent, tumor necrosis factor inhibitor, endothelininhibitor, angiotensin-converting enzyme inhibitor, calcium antagonist,antibiotic agent, antiviral agent or viral vector.

The present invention additionally provides a method of treating acardiovascular disease in a subject by administering to the subject anantibody, or antigen-binding fragment thereof, that selectively homes toheart vasculature and that specifically binds bc10 (SEQ ID NO: 35). Theinvention also provides a method of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to an antibody, or antigen-bindingfragment thereof, that selectively homes to heart vasculature and thatspecifically binds bc10 (SEQ ID NO: 35), thereby directing the moiety toheart vasculature. A method of the invention can be practiced, forexample, with a monoclonal antibody or an antibody with biologicalactivity. In the methods of the invention for directing a moiety toheart vasculature, any of a variety of moieties can be linked to anantibody that specifically binds bc10 (SEQ ID NO: 35). Such moietiesinclude, yet are not limited to, detectable moieties and therapeuticagents such as angiogenic agents, anti-thrombotic agents,anti-inflammatory agents, immunosuppressive agents, anti-arrhythmicagents, tumor necrosis factor inhibitors, endothelin inhibitors,angiotensin-converting enzyme inhibitors, calcium antagonists,antibiotic agents, antiviral agents and viral vectors.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as polypeptidefragments of antibodies that retain binding activity for a heart homingreceptor of at least about 1×10⁵ M⁻¹. One skilled in the art understandsthat antibodies that selectively home to heart vasculature, including,without limitation, Fab, F(ab′)₂ and Fv fragments, can retain bindingactivity for a heart homing receptor and, thus, are included within thedefinition of antibody. In addition, the term “antibody,” as usedherein, encompasses non-naturally occurring antibodies and fragmentsusually containing, at a minimum, one V_(H) and one V_(L) domain, suchas chimeric antibodies, humanized antibodies and single chain Fvfragments (scFv) that specifically bind a heart homing receptor. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, produced recombinantly or obtained, for example, byscreening phage-displayed or other combinatorial libraries such as thoseconsisting of variable heavy and light chains as described in Borrebaeck(Ed.), Antibody Engineering (Second edition) New York: Oxford UniversityPress (1995)) using, for example, an assay described herein below.

Antibodies which selectively home to heart vasculature also can beprepared, for example, using an HLP/CRIP2, receptor clone 9,Sigirr/TIR8, MpcII-3-related protein or bc10 fusion protein or asynthetic peptide encoding a portion of HLP/CRIP2, receptor clone 9,Sigirr/TIR8, MpcII-3-related protein or bc10 as an immunogen. Oneskilled in the art understands that purified human, murine or otherHLP/CRIP2, receptor clone 9, Sigirr/TIR8, MpcII-3-related protein orbc10, which can be produced recombinantly, for example, using thenucleic acid sequences disclosed herein as SEQ ID NO: 24, 26, 28, 32 and34, as well as fragments and peptide portions of HLP/CRIP2, receptorclone 9, Sigirr/TIR8, MpcII-3-related protein or bc10, can be useful asimmunogens for raising anti-HLP/CRIP2, anti-Sigirr/TIR8,anti-MpcII-3-related protein or anti-bc10 antibodies that selectivelyhome to heart vasculature. It is understood that fragments of HLP/CRIP2,receptor clone 9, Sigirr/TIR8, MpcII-3-related protein or bc10 useful asimmunogens include, without limitation, fragments that serve to produceantibodies. One skilled in the art further understands thatnon-immunogenic fragments or synthetic peptides of a heart homingreceptor can be made immunogenic by coupling the hapten to a carriermolecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). In addition, various other carrier molecules and methods forcoupling a hapten to a carrier molecule are well known in the art asdescribed, for example, by Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1988)).

Based on the identification of molecules that selectively home to heartvasculature and their ability to target a linked therapeutic agent toheart vasculature, the present invention provides methods for directinga moiety to heart vasculature and methods of treating a cardiovasculardisease. The present invention provides, for example, methods ofdirecting a moiety to heart vasculature in a subject by administering tothe subject a conjugate containing a moiety linked to a homing moleculethat selectively homes to heart vasculature and specifically bindsHLP/CRIP2 (SEQ ID NO: 25), thereby directing the moiety to heartvasculature. In a method of the invention, a homing molecule can home tothe heart in vivo with a selectivity, for example, of at least 5-foldrelative to non-recombinant phage, and can be, for example, a homingpeptide or peptidomimetic. In one embodiment, a method of the inventionfor directing a moiety to heart vasculature relies on a homing peptideor peptidomimetic containing the amino acid sequence CRPPR (SEQ IDNO: 1) or a conservative variant or peptidomimetic thereof. In anotherembodiment, a method of the invention relies on a homing peptide orpeptidomimetic that contains the amino acid sequence CGRKSKTVC (SEQ IDNO: 2) or a conservative variant or peptidomimetic thereof. A homingpeptide or peptidomimetic useful in the invention may optionally beconformationally constrained. Furthermore, any of a variety of moietiescan be useful in the methods of the invention including, withoutlimitation, detectable moieties such as radionuclides and fluorescentmolecules. Moieties useful in the invention further encompass, withoutlimitation, therapeutic agents such as angiogenic agents,anti-thrombotic agents, anti-inflammatory agents, immunosuppressiveagents, anti-arrhythmic agents, tumor necrosis factor inhibitors,endothelin inhibitors, angiotensin-converting enzyme inhibitors, calciumantagonists, antibiotic agents, antiviral agents and viral vectors.

The present invention further provides a method of directing a moiety toheart vasculature in a subject by administering to the subject aconjugate containing a moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically binds SEQ ID NO:27, thereby directing the moiety to heart vasculature. A homing moleculeuseful in the invention can home to the heart in vivo with aselectivity, for example, of at least 5-fold relative to non-recombinantphage and can be a homing peptide or peptidomimetic. It is understoodthat any of a variety of homing molecules that specifically bind SEQ IDNO: 27 can be useful for directing a moiety to heart vasculature. Suchhoming molecules include, but are not limited to, homing peptides andpeptidomimetics containing the amino acid sequence CARPAR (SEQ ID NO: 5)or a conservative variant or peptidomimetic thereof; and homing peptidesand peptidomimetics containing the amino acid sequence CPKRPR (SEQ IDNO: 6) or a conservative variant or peptidomimetic thereof. Moietiesuseful in the invention include, without limitation, detectable moietiessuch as radionuclides and fluorescent molecules as well as therapeuticagents such as angiogenic agents, anti-thrombotic agents,anti-inflammatory agents, immunosuppressive agents, anti-arrhythmicagents, tumor necrosis factor inhibitors, endothelin inhibitors,angiotensin-converting enzyme inhibitors, calcium antagonists,antibiotic agents, antiviral agents and viral vectors.

Further provided herein are methods of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatewhich contains a moiety linked to a homing molecule that selectivelyhomes to heart vasculature and specifically binds Sigirr/TIR8 (SEQ IDNO: 29), thereby directing the moiety to heart vasculature. As anon-limiting example, a homing molecule useful in a method of theinvention can home to the heart in vivo with a selectivity of at least5-fold relative to non-recombinant phage. As a further non-limitingexample, a homing molecule useful in a method of the invention can be ahoming peptide or peptidomimetic.

Any of a variety of homing molecules that specifically bind Sigirr/TIR8(SEQ ID NO: 29) are useful in the methods of the invention including,without limitation, homing peptides or peptidomimetics containing theamino acid sequence CKRAVR (SEQ ID NO: 7) or conservative variants orpeptidomimetics thereof; peptides and peptidomimetics containing theamino acid sequence CRNSWKPNC (SEQ ID NO: 8) or conservative variants orpeptidomimetics thereof; and peptides and peptidomimetics containing theamino acid sequence RGSSS (SEQ ID NO: 9) or conservative variants orpeptidomimetics thereof. Such homing peptides and peptidomimetics canbe, without limitation, linear, cyclic, or otherwise conformationallyconstrained. Furthermore, any of a variety of moieties can be useful inthe methods of the invention which rely on a homing molecule thatspecifically binds Sigirr/TIR8 (SEQ ID NO: 29). Such moieties include,without limitation, detectable moieties such as radionuclides andfluorescent molecules, and therapeutic agents such as angiogenic agents,anti-thrombotic agents, anti-inflammatory agents, immunosuppressiveagents, anti-arrhythmic agents, tumor necrosis factor inhibitors,endothelin inhibitors, angiotensin-converting enzyme inhibitors, calciumantagonists, antibiotic agents, antiviral agents and viral vectors.

The present invention further provides methods of directing a moiety toheart vasculature in a subject by administering to the subject aconjugate containing a moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically binds SEQ ID NO:33, thereby directing the moiety to heart vasculature. As non-limitingexamples, the methods of the invention can be practiced with a homingmolecule that homes to the heart in vivo with a selectivity of at least5-fold relative to non-recombinant phage, or with a homing moleculewhich is a peptide or peptidomimetic. In one embodiment, a method of theinvention is practiced with a conjugate containing a homing peptide orpeptidomimetic that includes the amino acid sequence CRSTRANPC (SEQ IDNO: 11) or a conservative variant or peptidomimetic thereof. In such amethod, the homing peptide or peptidomimetic containing the amino acidsequence CRSTRANPC (SEQ ID NO: 11) can optionally be conformationallyconstrained. Any of a variety of moieties can be useful in the methodsof the invention, including, without limitation, detectable moieties,such as radionuclides and fluorescent molecules, and therapeutic agentssuch as angiogenic agents, anti-thrombotic agents, anti-inflammatoryagents, immunosuppressive agents, anti-arrhythmic agents, tumor necrosisfactor inhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors.

Further provided herein are methods of directing a moiety to heartvasculature in a subject by administering to the subject a conjugatecontaining a moiety linked to a homing molecule that selectively homesto heart vasculature and specifically binds bc10 (SEQ ID NO: 35),thereby directing the moiety to heart vasculature. A homing moleculeuseful in the invention can home to the heart in vivo with aselectivity, for example, of at least 5-fold relative to non-recombinantphage and can be a peptide or peptidomimetic. Any of a variety of homingmolecules that specifically bind bc10 (SEQ ID NO: 35) can be useful inthe methods of the invention. Such homing molecules include, but are notlimited to, homing peptides and peptidomimetics containing the aminoacid sequence CPKTRRVPC (SEQ ID NO: 12) or a conservative variant orpeptidomimetic thereof, and homing peptides and peptidomimetics thatcontain the amino acid sequence CSGMARTKC (SEQ ID NO: 13) or aconservative variant or peptidomimetic thereof. Such homing peptides andpeptidomimetics can be useful as linear, cyclic or otherwiseconformationally constrained structures. The methods of the inventionwhich rely on a conjugate that incorporates a homing molecule whichspecifically binds bc10 (SEQ ID NO: 35) can be practiced with any of avariety of moieties. Useful moieties include, without limitation,detectable moieties such as radionuclides and fluorescent molecules aswell as therapeutic agents such as angiogenic agents, anti-thromboticagents, anti-inflammatory agents, immunosuppressive agents,anti-arrhythmic agents, tumor necrosis factor inhibitors, endothelininhibitors, angiotensin-converting enzyme inhibitors, calciumantagonists, antibiotic agents, antiviral agents and viral vectors.

The present invention also provides a method of treating acardiovascular disease in a subject by administering to the subject aconjugate containing a therapeutic agent linked to a homing moleculethat selectively homes to heart vasculature and specifically bindsHLP/CRIP2 (SEQ ID NO: 25). In such a method of the invention, a homingmolecule can home to the heart in vivo with a selectivity, for example,of at least 5-fold relative to non-recombinant phage, and can be, forexample, a homing peptide or peptidomimetic. Any of a variety of homingmolecules that specifically bind HLP/CRIP2 (SEQ ID NO: 25) can be usefulin a method of the invention including, without limitation, homingpeptides or peptidomimetics containing the amino acid sequence CRPPR(SEQ ID NO: 1) or conservative variants or peptidomimetics thereof, andhoming peptides or peptidomimetics containing the amino acid sequenceCGRKSKTVC (SEQ ID NO: 2) or conservative variants or peptidomimeticsthereof. A homing peptide or peptidomimetic useful in the invention mayoptionally be conformationally constrained. Any of a variety oftherapeutic agents can be useful for treating a cardiovascular diseaseaccording to a method of the invention, including, but not limited to,angiogenic agents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors.

Further provided herein are methods of treating a cardiovascular diseasein a subject by administering to the subject a conjugate containing atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature and specifically binds SEQ ID NO: 27. A homingmolecule useful for treating a cardiovascular disease can home to theheart in vivo with a selectivity, for example, of at least 5-foldrelative to non-recombinant phage. Any of a variety of homing moleculesthat specifically bind SEQ ID NO: 27 can be useful in a method of theinvention, including homing peptides or peptidomimetics such as thosecontaining the amino acid sequence CARPAR (SEQ ID NO: 5) or conservativevariants or peptidomimetics thereof, and those containing the amino acidsequence CPKRPR (SEQ ID NO: 6) or conservative variants orpeptidomimetics thereof. Any of a variety of therapeutic agents can beuseful for treating a cardiovascular disease according to a method ofthe invention. Such therapeutic agents include, but are not limited to,angiogenic agents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors.

Also provided herein are methods of treating a cardiovascular disease ina subject by administering to the subject a conjugate which contains atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature and specifically binds Sigirr/TIR8 (SEQ ID NO: 29). Ina method of the invention, the homing molecule can home to the heart invivo with a selectivity of, for example, at least 5-fold relative tonon-recombinant phage. Any of a variety of homing molecules thatspecifically bind Sigirr/TIR8 (SEQ ID NO: 29) are useful in the methodsof the invention including, but not limited to, homing peptides andpeptidomimetics. Such homing peptides or peptidomimetics can be, forexample, homing peptides and peptidomimetics having the amino acidsequence CKRAVR (SEQ ID NO: 7) or conservative variants orpeptidomimetics thereof; homing peptides and peptidomimetics having theamino acid sequence CRNSWKPNC (SEQ ID NO: 8) or conservative variants orpeptidomimetics thereof; or homing peptides and peptidomimetics havingpeptides and peptidomimetics having the amino acid sequence RGSSS (SEQID NO: 9) or conservative variants or peptidomimetics thereof. Homingpeptides and peptidomimetics useful in a method of the invention fortreating a cardiovascular disease can be, for example, linear, cyclic,or otherwise conformationally constrained. Any of a variety oftherapeutic agents can be linked to the homing molecule thatspecifically binds Sigirr/TIR8 (SEQ ID NO: 29) in a method of theinvention; such therapeutic agents include, without limitation,angiogenic agents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors.

The present invention further provides methods of treating acardiovascular disease in a subject by administering to the subject aconjugate containing a therapeutic agent linked to a homing moleculethat selectively homes to heart vasculature and specifically binds SEQID NO: 33. As non-limiting examples, such methods can be practiced witha homing molecule that homes to the heart in vivo with a selectivity ofat least 5-fold relative to non-recombinant phage, or with a homingmolecule which is a peptide or peptidomimetic. In particularembodiments, a method of the invention for treating a cardiovasculardisease is practiced using a homing peptide or peptidomimetic whichincludes the amino acid sequence CRSTRANPC (SEQ ID NO: 11) or aconservative variant or peptidomimetic thereof. Therapeutic agents whichcan be useful in a method of the invention include, without limitation,angiogenic agents, anti-thrombotic agents, anti-inflammatory agents,immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factorinhibitors, endothelin inhibitors, angiotensin-converting enzymeinhibitors, calcium antagonists, antibiotic agents, antiviral agents andviral vectors.

The present invention further provides methods of treating acardiovascular disease in a subject by administering to the subject aconjugate containing a therapeutic agent linked to a homing moleculethat selectively homes to heart vasculature and specifically bindsmurine bladder cancer-associated protein homolog (bc10; SEQ ID NO: 35).Homing molecules useful in the invention include, but are not limitedto, those which can home to the heart in vivo with a selectivity of atleast 5-fold relative to non-recombinant phage such as, withoutlimitation, homing peptides and peptidomimetics. Any of a variety ofhoming molecules that specifically bind bc10 (SEQ ID NO: 35) can beuseful for treating a cardiovascular disease according to a method ofthe invention. Such homing molecules include, but are not limited to,homing peptides and peptidomimetics containing the amino acid sequenceCPKTRRVPC (SEQ ID NO: 12) or a conservative variant or peptidomimeticthereof, and homing peptides and peptidomimetics containing the aminoacid sequence CSGMARTKC (SEQ ID NO: 13) or a conservative variant orpeptidomimetic thereof. As discussed above, such homing peptides andpeptidomimetics optionally can be conformationally constrained.Therapeutic agents suitable for use in a method of the inventioninclude, but are not limited to, angiogenic agents, anti-thromboticagents, anti-inflammatory agents, immunosuppressive agents,anti-arrhythmic agents, tumor necrosis factor inhibitors, endothelininhibitors, angiotensin-converting enzyme inhibitors, calciumantagonists, antibiotic agents, antiviral agents and viral vectors.

As discussed further below, the conjugates and methods of the inventioncan be useful for treating any of a variety of cardiopathies andcardiovascular diseases. Such cardiopathies and cardiovascular diseasesinclude, but are not limited to, coronary artery disease (CAD);atherosclerosis; thrombosis; restenosis; vasculitis including autoimmuneand viral vasculitis such as polyarteritis nodosa, Churg-Strasssyndrome, Takayasu's arteritis, Kawasaki Disease and Rickettsialvasculitis; atherosclerotic aneurisms; myocardial hypertrophy;congenital heart diseases (CHD); ischemic heart disease and anginas;acquired valvular/endocardial diseases; primary myocardial diseasesincluding myocarditis; arrhythmias; and transplant rejection.Cardiopathies and cardiovascular diseases to be treated according to amethod of the invention further include, but are not limited to,metabolic myocardial diseases and myocardiomyopathies such ascongestive, hypertrophic and restrictive cardiomyopathies, and hearttransplants. A therapeutic agent linked to a homing molecule of theinvention will concentrate in the heart blood vessels and can furtheraccumulate in the myocardium. Thus, the conjugates and methods of theinvention are useful for treating these and other disorders of heartblood vessels or myocardium.

Angiogenesis-based therapy using a therapeutic agent that stimulates newblood vessel formation (angiogenesis) can be useful for treating acardiovascular disease according to a method of the invention.Angiogenic agents can be useful for treating, without limitation,ischemic heart disease including chronic myocardial ischemia and acutemyocardial infarction (Ware and Simons, Nature Med. 3:158-164 (1997)).Many patients with severe vascular disease that are not candidates formechanical revascularization can benefit from angiogenesis-basedtherapy, including those patients with occlusion of vessels too small tobe bypassed, those without conduits and those who are not surgicalcandidates because of concomitant disease. It has been calculated that314 million disease cases in the U.S. and European Union may benefitfrom angiogenesis-based therapy (Miller and Abrams, Gen. Engin. News18:1 (1998)). Thus, a molecule that selectively homes to heartvasculature can be linked to an angiogenic agent and delivered to apatient, thereby stimulating angiogenesis and alleviating thecardiovascular disease.

An angiogenic agent useful in the invention also can be a naturallyoccurring angiogenic growth factor or cytokine that induces or promotesangiogenesis by stimulating endothelial cell growth or migration.Angiogenic agents useful in the invention encompass, without limitation,isoforms of vascular endothelial growth factor (VEGF) such as VEGF-A,including VEGF₁₂₁ and VEGF₁₆₅, and forms of fibroblast growth factorincluding, but not limited to, forms of FGF-I and FGF-2 (Ruel andSellke, Sem. Thor. Cardiovasc. Sur. 15:222-235 (2003). As discussedfurther below, angiogenic agents and other therapeutic agents of theinvention can be delivered as protein therapeutics or as nucleic acidtherapeutic via gene therapy vectors.

VEGF-A, which is additionally known as vascular permeability factor(VPF), also can be useful in the invention (Dvorak et al., Am. J.Pathol. 146:1029-1039 (1995); Thomas et al., J. Biol. Chem. 271:603-606(1996); Olofsson et al., Proc. Natl. Acad. Sci., USA 93:2576-2581(1996); Joukov et al., EMBO J. 15:290-298 (1996); and Harada et al., Am.J. Physiol. 270:H1791-H1802 (1996)). Other VEGFs (B, C and D) haveangiogenic activity without effecting endothelial permeability. Transferof plasmid DNA encoding VEGF has been shown to result in significantreduction in thrombus formation and intimal thickening following stentimplantation (J. Am. Coll. Cardiol. 29:1371-1379 (1997)). Angiogenicagents useful in the invention include, without limitation, arecombinant 165 kDa isoform of VEGF, designated rhVEGF, developed byGenentech; a nucleic acid molecule encoding the 121 amino acid isoformof VEGF (BIOBYPASS™; GenVec/Parke Davis); and nucleic acids encodingVEGF-B, VEGF-C and VEGF-D. See, for example, Miller and Abrams, supra,1998. An angiogenic agent useful in the invention also can be a memberof the fibroblast growth factor (FGF) family such as FGF-1 (acidic),FGF-2 (basic), FGF-4 or FGF-5 (Slavin et al., Cell Biol. Int. 19:431-444(1995); Folkman and Shing, J. Biol. Chem. 267:10931-10934 (1992)). AnFGF to be linked to a molecule that selectively homes to heartvasculature in a conjugate or method of the invention can be, forexample, FIBLAST®-(trafermin), a recombinant form of FGF-2 beingdeveloped by Scios, Inc. (Mountain View, Calif.) and Wyeth AyerstLaboratories (Radnor, Pa.), or GENERX™, or an adenoviral gene therapyvector encoding FGF-4 developed by Collateral Therapeutics (San Diego,Calif.) and Schering AG (Miller and Abrams, supra, 1998).

An angiogenic agent useful in the invention also can be angiopoietin-1,an angiogenic factor that signals through the endothelial cell-specificTie2 receptor tyrosine kinase (Davis et al., Cell 87:1161-1169 (1996)).Like VEGF, angiopoietin-1 is essential for normal vascular development,and its overexpression leads to increased angiogenesis (Suri et al.,Cell 87:1171-1180 (1996)).

A therapeutic agent useful in a conjugate or method of the inventionalso can be an anti-thrombotic agent that prevents the formation of athrombus, which is an aggregation of blood factors, primarily plateletsand fibrin with entrapment of cellular elements. Thrombus formation isstimulated by the presence of atheromatous plaques and is the main causeof episodes of acute ischemic heart disease. An anti-thrombotic agentuseful in the invention can be, without limitation, an inhibitor of theIIb/IIIa integrin (Coller, Circulation 92:2373 (1995); a tissue factorinhibitor; a plasminogen activator or an anti-thrombin agent.

An immunosuppressive agent also can be useful in the conjugates andmethods of the invention, for example, for treating a heart transplantrecipient. It is understood that an immunosuppressive agent can beuseful in chronic prophylactic treatment, which organ transplantrecipients typically require for their entire lives, or for treatingpatients exhibiting one or more symptoms consistent with transplantrejection, or for use as a rescue agent in severe rejection.Immunosuppressive agents useful in the conjugates and methods of theinvention encompass, without limitation, steroids includingcorticosteroids and prednisolone; calcineurin inhibitors such asPROGRAF®, NEORAL1®, RAPAMUNE®, cyclosporine A and other cyclosporines;anti-proliferative agents including CELLCEPT®, IMURAN® (azathioprine)and CERTICAN™ (everolimus); and therapeutic monoclonal antibodies suchas OKT3, ATGAM, thymoglobulin and anti-thymocyte globulins, dicluzimaband basiliximab. These and other immunosuppressive agents can be usefulalone or in combination with another immunosuppressive agent or othertherapeutic agent in the conjugates and methods of the invention.

Anti-inflammatory agents, which are molecules that reduce one or moresymptoms of inflammation, also are useful in the conjugates and methodsof the invention. Such anti-inflammatory agents include, withoutlimitation, steroids including corticosteroids and immunoglobulins aswell as cyclooxygenase inhibitors and other non-steroidalanti-inflammatory drugs. In one embodiment, the anti-inflammatory agentis AGI-1067, a cholesterol-lowering anti-inflammatory agent(AtheroGenics; Atlanta, Ala.).

An anti-arrhythmic agent also can be useful in the conjugates andmethods of the invention, for example, for treating a cardiacarrhythmia, which is a disorder in which the normal periodicity andregular electromechanical activity of the heart are disrupted. Thus, ina conjugate or method of the invention, a therapeutic agent can be anantiarrhythmic agent such as a local anesthetic (class I agent),sympathetic antagonist (class II agent), antifibrillatory agent (classIII agent), calcium channel agent (class IV agent) or anion antagonist(class V agent) as described, for example, in Vukmir, Am. J. Emer. Med.13:459-470 (1995); Grant, PACE 20:432-444 (1997); Mann, Curr. Med. Res.Opin. 13:325-343 (1995); or Lipka et al., Am. Heart J. 130:632-640(1995). An antiarrhythmic agent that is a local anesthetic acts as afast sodium channel antagonist; such an antiarrhythmic agent can be,without limitation, procainamide, quinidine or disopyramide; lidocaine,phenyloin, tocamide or mexiletine; or encamide; flecamide; lorcamide;propafenone (III) or moricizine (Vukmir, supra, 1995). An antiarrhythmicagent also can be sympathetic antagonist (β-adrenergic antagonist) suchas, without limitation, propranolol, esmolol, metoprolol, atenelal,acebutolol, or an antifibrillatory agent that acts by prolonging actionpotential duration (APD), for example, bretylium, amiodarone, sotalol(II) or N-acetylprocainamide (Vukmir, supra, 1995). Additionalantiarrhythmic agents that can be useful as therapeutic agents in theconjugates or methods of the invention include, but are not limited to,calcium channel agents such as verapamil, diltiazem, and bepridil, andanion antagonists such as alinidine (Vukmir, supra, 1995). The skilledperson understands that these and other antiarrhythmic drugs known inthe art can be therapeutic agents which are useful for treating cardiacarrhythmias in a conjugate or method of the invention. Calciumantagonists, also known as calcium channel blockers (CBBs), havebeneficial effects in many cardiovascular diseases, acting as potentinhibitors of smooth muscle cell proliferation and migration. Additionalproperties that make these therapeutic agents useful in treatingatherosclerosis and other cardiovascular disease include their abilityto inhibit calcium influx into the vascular wall; reduce extracellularmatrix synthesis; promote uptake and breakdown of low-densitylipoproteins; protect lipoproteins from oxidative modification; maintainendothelial cell function; and inhibit platelet activation. Among thecalcium antagonists, amlodipine is a therapeutic agent with vascularselectivity (Marche et al., Int. J. Cardiol. 62 (Suppl.): S17-S22(1997); Schachter, Int. J. Cardiol. 62 (Suppl.): S85-S90 (1997)).Additional calcium antagonists which can serve as therapeutic agentsuseful in the invention include, without limitation, nicardipine,nifedipine, propanolol, isosorbide dinitrate, diltiazem, and isradipine(Nayler (Ed.) Calcium Antagonists pages 157-260 London: Academic Press(1988); Schachter, Int. J. Cardiol. 62 (Suppl.): S9-S15 (1997)).Therapeutic agents that elevate cAMP or cGMP content reduce vascularsmooth muscle cell responsiveness or proliferation and also can beuseful when linked to a molecule that selectively homes to heartvasculature. Such therapeutic agents include, yet are not limited to,the cAMP phosphodiesterase inhibitor cilostasol and endothelium-derivednitric oxide (NO) or NO-generating vasodilators. See, for example,Takahashi et al., J. Cardiovas. Pharm. 20:900-906 (1992); and Cornwellet al., Am. J. Physiol. 267:C1405-1413 (1994).

A therapeutic agent useful in the invention also can be an antiviral orantibiotic agent. Numerous studies have reported an association ofendocarditis, atherosclerosis and restenosis with particular bacterialand viral infections, especially cytomegalovirus and Chlamydiapneumoniae (Cheng and Rivera, Annals of Pharmacotheraipy 32:1310-1316(1998)). For prophylactic use in a heart transplant or other patient athigh risk of developing atherosclerosis, the patient can be administereda conjugate containing a molecule that selectively homes to heartvasculature linked to an antiviral agent such as ganciclovir. Additionalantiviral agents that can be included in a conjugate or method of theinvention include, without limitation, Ribavirin (Virazole,1-β-D-ribofuranosyl-1,2,4-triazole-3-carboximide) and recombinant humanleukocyte IFN-α A/D (Matsumori et al., Circulation 71:834-839 (1985);Matsumori et al., J. Am. Coll. Cardiol. 9:1320-1325 (1987)).

The conjugates and methods also can be useful for treatingatherosclerosis, which, in conjunction with its consequences,constitutes the most common and important cause of disease and death inthe western world. Like other occlusive vascular disease,atherosclerosis is characterized by the abnormal accumulation of lipid,inflammatory cells, vascular smooth muscle cells, and extracellularmatrix proteins within the intimal space between the endothelial liningand the medial layer (plaque formation). In particular, damage to theendothelium allows entry of cholesterol-rich low-density lipoproteins(LDLs) into the intima. Lipid is taken up by macrophages in the intima,with excessive lipid accumulating in the intimal macrophages through areceptor-independent pathway that takes up oxidized LDL. Macrophagesrelease lipid into the intima and secrete cytokines that stimulateproliferation. Intimal cells with features of myofibroblasts secretecollagen, causing the plaque to become fibrotic in some cases. As thelesion develops, there is pressure atrophy of the media, and the elasticlamina is disrupted. Further collagen deposition forms a dense fibrouscap to the plaque (fibrolipid plaque), which contains free lipid as wellas lipid in macrophages. The fragile endothelium of the plaques oftenulcerates, allowing platelet aggregation and thrombosis. Growth factorssuch as platelet-derived growth factor cause further plaque developmentby stimulating cell proliferation.

Increased proliferation of intimal smooth muscle cells causes myointimalhyperplasia and luminal narrowing. The abnormal cell proliferation whichplays a role in neointima formation can result from specific growthfactors such as platelet derived growth factor (PDGF), transforminggrowth factor-β1 (TGF-β1) or angiotensin II (Ross, Annu. Rev. Physiol.57:791 (1995); Schwartz et al., Circ. Res. 77:445 (1995); and Gibbonsand Dzau, New Eng. J. Med. 330:1431 (1994)).

A therapeutic agent useful in the conjugates and methods of theinvention for treating a cardiovascular disease such as intimalhyperplasia following angioplasty can be a growth inhibitory agent thatreduces or prevents vascular disease by limiting neointimal smoothmuscle cell proliferation. For example, a herpes virus thymidine kinase(tk) gene and systemic ganciclovir can be used to kill proliferatingcells and limit neointimal formation. In one study, porcine iliofemoralarteries were infected with an adenoviral vector encoding tk and, afterexposure to ganciclovir, the neointimal thickening seen followingballoon injury was reduced by 50-87% (Ohno et al., Science 265:781-784(1994); see, also, Guzman et al., Proc. Natl. Acad. Sci. USA91:10732-10736 (1994); Chang et al., Mol. Med. 1:172-181 (1995); andSimari et al., Circulation 92:1-501 (1995)). Thus, a therapeutic agentuseful in the invention can be a cytostatic agent such as, withoutlimitation, thymidine kinase combined with ganciclovir.

Additional therapeutic agents for limiting neointimal formation also areknown in art and include genes that inhibit cell cycle proteins andproto-oncogenes (Simari and Nabel, Semin. Intervent. Cardiol. 1:77-83(1996)). Such therapeutic agents include, without limitation, tumorsuppressor genes such as p53 and retinoblastoma as well as genesencoding bcl-x, mutant forms of ras and nitric oxide synthetase. As anon-limiting example, the retinoblastoma gene product (Rb) inhibits cellproliferation in many mammalian cell types, and transfer of arecombinant adenovirus encoding active Rb into injured rat carotid andporcine iliac arteries decreases neointimal formation (Chang et al.,Science 267:518-522 (1994)). Gene transfer of p53 similarly has beenshown to inhibit vascular smooth muscle cell proliferation (Yonemitsu etal., Circ. Res. 82:147-156 (1998); see, also, Muller, Prog. Cardiovas.Dis. 40:117-128 (1997)). Direct gene transfer of dominant negative rasvariants also inhibited intima development following balloon injury inrat carotid and porcine iliofemoral models (Chang et al., J. Clin.Invest. 96:2260-2268 (1995)). In a rat carotid injury model, genetransfer of nitric oxide synthetase also limited intimal formation (vonder Leyen et al., Proc. Natl. Acad. Sci., USA 92:1137-1141 (1995). Oneskilled in the art understands that a cytostatic agents also can beuseful as a therapeutic agent in a conjugate or method of the invention.

A decoy or antisense oligonucleotide against a cellular target such asE2F or one of various cyclins also can be a therapeutic agent useful forlimiting neointima proliferation in a method of the invention (Morishitaet al., Circ. Res. 82:1023-1028 (1998); and Mann et al., Circulation96:1-4 1997)). The growth arrest homeobox gene gax, which is rapidlydownregulated in vascular smooth muscle cells after vascular injury invivo also can be a therapeutic agent useful for inhibiting intimalhyperplasia (Smith et al., Genes Dev. 11:1674-1689 (1997); and Skopickiet al., Circ. Res. 80:452-462 (1997)). Additional therapeutic agentsthat can be useful in limiting intimal hyperplasia in a conjugate ormethod of the invention also are known in the art (see, for example,Laitinen and Yla-Herttuala, Current Opin. Lipid. 9:465-469 (1998).

A conjugate or method of the invention also can be useful for treatingrestenosis, which is the re-narrowing of lumen dimensions that mayfollow angioplasty, a procedure in which a balloon is inserted into anoccluded vessel and then inflated to dilate the area of narrowing.Restenosis occurs in about 30 to 50% of cases over a time course ofthree to six months and involves cellular hyperplasia within theneointima, the organization of thrombus within the vessel wall, andshrinkage of overall vessel dimensions. Angioplasty denudes the vesselof endothelial cells that would normally generate paracrine inhibitorsof vascular smooth muscle migration and proliferation.

Nitric oxide deficiency is associated with impaired vasorelaxation andincreased adhesiveness, predisposing vascular tissue to the formation ofatherosclerotic lesions (Gibbons and Dzau, Science 272:689-693 (1996)).Therapeutic agents useful in the conjugates and methods of the inventioninclude, without limitation, endothelial cell-type nitric oxide (NO)synthase agents, which are agents that enhance nitric oxide synthaseactivity. Such therapeutic agents which enhance nitric oxide (NO)activity encompass, without limitation, nitric oxide synthase genes andnitric oxide-donor drugs. Transfection of a nitric oxide synthase geneinto the vessel wall after balloon injury in a rat model of neointimaformation resulted in generation of nitric oxide and a substantialinhibition of cell proliferation, migration and matrix productionrequired for neointima formation (von der Leyen et al., supra, 1995).Clinical studies also indicate that a nitric oxide-donor drug can beused to augment nitric oxide activity and treat restenosis (Ferguson,Circulation 90:4 (1994)).

The conjugates and methods of the invention also can be used to treatcongestive heart failure (CHF), a disorder affecting nearly five millionpeople in the United States alone. Congestive heart failure results whenthe heart is damaged from atherosclerosis or other conditions such ashigh blood pressure, myocardial infarction or defective heart valves.The failing heart works inefficiently, causing fluid retention,shortness of breath and fatigue. Thus, for treatment of congestive heartfailure, a molecule that selectively homes to heart vasculature can belinked to a therapeutic agent such as, without limitation, a TNFinhibitor such as the recombinant soluble TNF decoy receptor, EMBREL™(Immunex Corp.; Seattle, Wash.).

Endothelins, which are polypeptides that constrict blood vessels, alsoare elevated in congestive heart failure. Thus, a molecule of theinvention which selectively homes to heart vasculature can be linked toa therapeutic agent that is an endothelin inhibitor, for example, thesmall molecule drug, TBC11251. This drug, which was developed by TexasBiotechnology Corp., inhibits endothelin A-receptor binding and promotesrelaxation of smooth muscle cells. TBC11251 has resulted in significantimprovement in patients with moderate to severe congestive heart failure(Potera, Gen. Engin. News 18:12 (1998)).

A therapeutic agent for use in a conjugate or method of the inventionalso can be a replacement gene therapy vector for an inherited heartdisease. Such an inherited disease can be, for example, an inheriteddisease of the myocardium such as X-linked dilated cardiomyopathy,hypertrophic cardiomyopathy, Long QT Syndrome or another disease forwhich the disease-causing mutation has been identified (Bowles et al.,Cardiovas. Res. 35:422-430 (1997)). In one embodiment, a conjugate ormethod of the invention is practiced with a molecule that selectivelyhomes to heart vasculature linked to an adenoviral gene therapy vectorcontaining a nucleic acid molecule encoding a desired replacement geneproduct.

A therapeutic agent can be delivered, for example, as a protein asdescribed in Harada et al., J. Clin. Invest. 94:623-630 (1994).Microspheres, for example, microspheres of 7 μm diameter to which anangiogenic factor such as bFGF is reversibly adsorbed through SO₃residues, also can be linked to a molecule that selectively homes toheart vasculature in order to selectively deliver the microspheres fortreatment of a cardiovascular disease. Such microspheres lodge in theperipheral microcirculation without interfering with total flow and areslowly released over a period of a week (Arras et al., Nature Biotech.16:159-162 (1998); see, also, Tice and Staas, Nature Biotech. 16:134(1998)). A single injection of a biodegradable microsphere can be usedto deliver an angiogenic or other therapeutic agent, which is releasedover one or several months following the injection, with the rate andduration of drug release controlled by factors such as the polymer typeand microparticle size (Maulding, Controlled Release 6:167-176 (1987);Tice and Tabibi, pages 315-339 in Kydonieus (Ed.), Treatise onControlled Drug Delivery Marcel Dekker, New York (1992)). A gene therapyvector also can be linked to a homing molecule that selectively homes toheart vasculature to deliver a nucleic acid molecule encoding anangiogenic or other therapeutic agent (see, for example, Isner et al.,Lancet 348:370-374 (1996); Giordano et al., Nature Med. 2:534-539(1996))

A variety of suitable gene therapy methodologies are known in the artfor treating a cardiopathy or cardiovascular disease in a method of theinvention (see, for example, Li et al., Cardiol. 41:39-46 (1997)). Asnon-limiting examples, viral vectors, including retroviral andadenoviral vectors, can be useful for gene delivery when linked to amolecule that selectively homes to heart vasculature. Such a genetherapy vector can include a nucleic acid molecule encoding atherapeutic agent such as, without limitation, an angiogenic agent suchas a VEGF or FGF. Gene therapy with forms of VEGF, FGF and otherangiogenic agents are well known in the art as described, for example,in Sylven, Drugs of Today 38:819-827 (2002); Symes, J. Card. Surg.15:283-290 (2000); and Grines et al., Am. J. Cardiol. 92 (Suppl.):24N-31N (2003). Angiogenic gene therapy with a replication-incompetent,serotype 5 adenovirus in which the E1A and E1B genes have been replacedby the human fibroblast growth factor-4 (FGF-4) gene under control of acytomegalovirus promoter, denoted Ad5FGF-4, has had beneficial effectsin placebo-controlled trials in humans with chronic stable angina(Grines et al., supra, 2003). See, also, Simari and Nabel, supra, 1996;and Indolfi and Chiariello, Cardiologia 43:365-373 (1998)). Thus, in oneembodiment, a method of the invention relies on a replication deficientrecombinant adenoviral vector which expresses a therapeutic geneproduct.

In view of the above, it is understood that a variety of therapeuticagents are useful for treating a cardiovascular disease according to amethod of the invention. Useful therapeutic agents encompass, yet arenot limited to, angiogenic agents, anti-thrombotic agents,anti-inflammatory agents, immunosuppressive agents, anti-arrhythmicagents, tumor necrosis factor inhibitors, endothelin inhibitors,angiotensin-converting enzyme inhibitors, calcium antagonists,antibiotic agents, antiviral agents and viral vectors. One skilled inthe art understands that these as well as additional known or othertherapeutic agents can be selectively directed to heart vasculature whenincorporated into a conjugate or method of the invention. Furthermore,one skilled in the art of medicinal cardiology understands that theseand other therapeutic agents can be used separately or together in theconjugates and methods of the invention. It further is understood that aconjugate of the invention can contain one or more of such therapeuticagents and that additional components can optionally be included in aconjugate of the invention. As an example, in some cases, it can bedesirable to utilize an oligopeptide spacer between the homing moleculeand the therapeutic agent. See, for example, Fitzpatrick and Garnett,Anticancer Drug Design 10:1-9 (1995).

It is further understood that a variety of routes of administration areuseful in the methods of the invention. Such routes encompass systemicand local administration and include, without limitation, oraladministration, intravenous injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, transdermal diffusionor electrophoresis, local injection, and extended release deliverydevices including locally implanted extended release devices such asbioerodible or reservoir-based implants.

The present invention further provides methods of imaging heartvasculature in a subject by administering to the subject a conjugatecontaining a detectable moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically binds HLP/CRIP2(SEQ ID NO: 25); and detecting the conjugate. In the methods of theinvention for imaging heart vasculature, a homing molecule can home tothe heart in vivo with a selectivity, for example, of at least 5-foldrelative to non-recombinant phage, and can be, for example, a homingpeptide or peptidomimetic. A variety of homing molecules thatspecifically bind HLP/CRIP2 (SEQ ID NO: 25) can be useful for imagingheart vasculature according to a method of the invention. Such homingmolecules encompass, without limitation, homing peptides orpeptidomimetics containing the amino acid sequence CRPPR (SEQ ID NO: 1)or conservative variants or peptidomimetics thereof, and homing peptidesor peptidomimetics containing the amino acid sequence CGRKSKTVC (SEQ IDNO: 2) or conservative variants or peptidomimetics thereof. A variety ofdetectable moieties are useful in the above methods of the invention,including, for example, radionuclides and paramagnetic ions.

Further provided herein are methods of imaging heart vasculature in asubject by administering to the subject a conjugate containing adetectable moiety linked to a homing molecule that selectively homes toheart vasculature and that specifically binds SEQ ID NO: 27; anddetecting the conjugate. In the methods of the invention for imagingheart vasculature, a homing molecule can home to the heart in vivo witha selectivity, for example, of at least 5-fold relative tonon-recombinant phage. A variety of homing molecules that specificallybind SEQ ID NO: 27 can be useful for imaging heart vasculature accordingto a method of the invention. Such homing molecules encompass, withoutlimitation, homing peptides or peptidomimetics such as those containingthe amino acid sequence CARPAR (SEQ ID NO: 5) or conservative variantsor peptidomimetics thereof, and those containing the amino acid sequenceCPKRPR (SEQ ID NO: 6) or conservative variants or peptidomimeticsthereof. A variety of detectable moieties are useful for imaging heartvasculature including, for example, radionuclides and paramagnetic ions.

The present invention additionally provides methods of imaging heartvasculature in a subject by administering to the subject a conjugatewhich contains a detectable moiety linked to a homing molecule thatselectively homes to heart vasculature and specifically bindsSigirr/TIR8 (SEQ ID NO: 29); and detecting the conjugate. In suchimaging methods of the invention, the homing molecule can home to theheart in vivo with a selectivity of, for example, at least 5-foldrelative to non-recombinant phage. Furthermore, any of a variety ofhoming molecules that specifically bind Sigirr/TIR8 (SEQ ID NO: 29) areuseful for imaging heart vasculature according to a method of theinvention; such homing molecules include, without limitation, homingpeptides and peptidomimetics having the amino acid sequence CKRAVR (SEQID NO: 7) or conservative variants or peptidomimetics thereof; homingpeptides and peptidomimetics having the amino acid sequence CRNSWKPNC(SEQ ID NO: 8) or conservative variants or peptidomimetics thereof; andhoming peptides and peptidomimetics having the amino acid sequence RGSSS(SEQ ID NO: 9) or conservative variants or peptidomimetics thereof. Itis recognized that the homing peptides and peptidomimetics useful in theimaging methods of the invention can be, for example, linear, cyclic, orotherwise conformationally constrained. Detectable moieties useful forimaging include, but are not limited to, radionuclides and paramagneticions.

Further provided herein is a method of imaging heart vasculature in asubject by administering to the subject a conjugate containing adetectable moiety linked to a homing molecule that selectively homes toheart vasculature and specifically binds SEQ ID NO: 33; and detectingthe conjugate. As non-limiting examples, the imaging methods of theinvention can be practiced with a homing molecule that homes to theheart in vivo with a selectivity of at least 5-fold relative tonon-recombinant phage, or with a homing molecule which is a peptide orpeptidomimetic. Homing molecules useful in the imaging methods of theinvention include, without limitation, homing peptides andpeptidomimetics such as those having the amino acid sequence CRSTRANPC(SEQ ID NO: 11) or a conservative variant or peptidomimetic thereof. Themethods of the invention for imaging heart vasculature can be practicedwith any of a variety of detectable moieties including, for example,radionuclides and paramagnetic ions.

Also provided herein is a method of imaging heart vasculature in asubject by administering to the subject a conjugate containing adetectable moiety linked to a homing molecule that selectively homes toheart vasculature and specifically binds murine bladdercancer-associated protein homolog (bc10; SEQ ID NO: 35); and detectingthe conjugate. Homing molecules useful for imaging according to a methodof the invention include, but are not limited to, those which can hometo the heart in vivo with a selectivity of at least 5-fold relative tonon-recombinant phage. Any of a variety of homing molecules thatspecifically bind bc10 (SEQ ID NO: 35) can be useful for imaging heartvasculature according to a method of the invention, including homingpeptides and peptidomimetics such as those containing the amino acidsequence CPKTRRVPC (SEQ ID NO: 12) or a conservative variant orpeptidomimetic thereof, or those containing the amino acid sequenceCSGMARTKC (SEQ ID NO: 13) or a conservative variant or peptidomimeticthereof. Homing peptides and peptidomimetics useful in the invention canbe utilized in linear, cyclic or otherwise conformationally constrainedform. As indicated above, detectable moieties useful for imaginginclude, yet are not limited to, radionuclides and paramagnetic ions.

The imaging methods of the invention rely on a detectable moiety. Asused herein, the term “detectable moiety” refers to any molecule whichcan be administered in vivo and subsequently detected. Exemplarydetectable moieties useful in the conjugates and imaging methods of theinvention include paramagnetic ions, radionuclides and fluorescentmolecules. Exemplary radionuclides include indium-11, technetium-99,carbon-11, and carbon-13. Fluorescent molecules include, withoutlimitation, fluorescein, allophycocyanin, phycoerythrin, rhodamine, andTexas red. Where a detectable moiety is a gamma ray emittingradionuclide such as indium-113, indium-115 or technetium-99, theconjugate can be visualized using a solid scintillation detectorfollowing administration to the subject.

Also provided herein is a method of isolating one or more homingmolecules that selectively homes to heart vasculature by contactingHLP/CRIP2, or a fragment thereof, with a library of molecules underconditions suitable for specific binding of a molecule to HLP/CRIP2;assaying for specific binding; and separating one or moreHLP/CRIP2-binding molecules from the library, thereby isolating one ormore homing molecules that selectively homes to heart vasculature andspecifically binds HLP/CRIP2. Cells that express HLP/CRIP2 on the cellsurface as well as purified HLP/CRIP2, or a fragment thereof, can beuseful in the screening methods of the invention. As non-limitingexamples, native, recombinant, human, murine (SEQ ID NO: 25) and othervariants and homologs of HLP/CRIP2, and fragments thereof such as,without limitation, CRPPR (SEQ ID NO: 1) or CGRKSKTVC (SEQ ID NO:2)-binding fragments of HLP/CRIP2, whether purified or expressed on thesurface of a cell, can be useful in the screening methods of theinvention. Libraries that can be screened according to a method of theinvention include, but are not limited to, libraries of peptides andpeptidomimetics, libraries of small molecules, and libraries ofantibodies and antigen-binding fragments thereof, including synthetic,single-chain or other antibody libraries. In one embodiment, a method ofthe invention includes a further step of assaying for localization ofone or more of the separated molecules of the library subsequent tointravenous injection. Where a fragment of HLP/CRIP2 is utilized inplace of full-length HLP/CRIP2, it is understood that such a fragmentcan be, without limitation, a fragment which specifically binds SEQ IDNO: 1 or SEQ ID NO: 2.

Any of a variety of HLP/CRIP2 polypeptides can be useful in the methodsof the invention for isolating molecules that selectively home to heartvasculature. As used herein, the term “HLP/CRIP2” means a polypeptidewhich is selectively expressed in heart vasculature and which has atleast 60% amino acid identity with SEQ ID NO: 25. In particularembodiments, HLP/CRIP2 has at least 70%, 80%, 90%, 95% or more aminoacid identity with SEQ ID NO: 25.

Further provided by the present invention is a method of isolating oneor more homing molecules that selectively homes to heart vasculature bycontacting receptor clone 9, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto receptor clone 9; assaying for specific binding; and separating oneor more receptor clone 9-binding molecules from the library, therebyisolating one or more homing molecules that selectively homes to heartvasculature and specifically binds receptor clone 9. The methods of theinvention can be practiced with cells that express receptor clone 9 onthe cell surface as well as purified receptor clone 9 or a fragmentthereof. As non-limiting examples, native, recombinant, human, murine(SEQ ID NO: 27) and other variants and homologs of murine receptor clone9 (SEQ ID NO: 27), and fragments thereof such as, without limitation,SEQ ID NO: 5 and SEQ ID NO: 6-binding fragments of receptor clone 9,whether purified or expressed on the surface of a cell, can be useful inthe screening methods of the invention. Any of a variety of librariescan be screened according to a method of the invention including, butnot limited to, those described hereinabove.

As used herein, the term “receptor clone 9” means a polypeptide which isselectively expressed in heart vasculature and which has at least 60%amino acid identity with SEQ ID NO: 27. In particular embodiments,receptor clone 9 has at least 70%, 80%, 90%, 95% or more amino acididentity with SEQ ID NO: 27.

The present invention also provides a method of isolating one or morehoming molecules that selectively homes to heart vasculature bycontacting Sigirr/TIR8, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto Sigirr/TIR8; assaying for specific binding; and separating one ormore Sigirr/TIR8-binding molecules from the library, thereby isolatingone or more homing molecules that selectively homes to heart vasculatureand specifically binds Sigirr/TIR8. Cells that express Sigirr/TIR8 onthe cell surface as well as purified Sigirr/TIR8, or a fragment thereof,can be useful in the screening methods of the invention. As non-limitingexamples, native, recombinant, human, murine (SEQ ID NO: 29) and othervariants and species homologs of Sigirr/TIR8, as well as fragmentsthereof such, without limitation, SEQ ID NO: 7 or SEQ ID NO: 8-bindingfragments, whether purified or expressed on the surface of a cell, canbe useful in the screening methods of the invention. It is furtherunderstood that, as indicated above, any of a variety of libraries canbe useful in the screening methods of the invention.

The term “Sigirr/TIR8,” as used herein, means a polypeptide which isselectively expressed in heart vasculature and which has at least 60%amino acid identity with SEQ ID NO: 29. In particular embodiments,Sigirr/TIR8 has at least 70%, 80%, 90%, 95% or more amino acid identitywith SEQ ID NO: 29.

Further provided herein is a method of isolating one or more homingmolecules that selectively homes to heart vasculature by contactingMpcII-3-related protein, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto MpcII-3-related protein; assaying for specific binding; andseparating one or more MpcII-3-related protein-binding molecules fromthe library, thereby isolating one or more homing molecules thatselectively homes to heart vasculature and specifically bindsMpcII-3-related protein. A screening method of the invention can bepracticed, for example, with cells that express MpcII-3-related proteinon the cell surface or with purified MpcII-3-related protein or afragment thereof. As non-limiting examples, native, recombinant, human,murine (SEQ ID NO: 33) and other variants and species homologs ofMpcII-3-related protein, as well as fragments of SEQ ID NO: 33 or humanor other MpcII-3-related proteins, including, without limitation,CRSTRANPC (SEQ ID NO: 11)-binding fragments, whether purified orexpressed on the surface of a cell, can be useful in the screeningmethods of the invention. The methods of the invention can be used toscreen any of a variety of libraries such as, without limitation,libraries of peptides and peptidomimetics, libraries of small molecules,and libraries of antibodies or antigen-binding fragments thereof,including synthetic, single-chain or other antibody libraries.

As used herein, the term “MpcII-3-related protein” means a polypeptidewhich is selectively expressed in heart vasculature and which has atleast 60% amino acid identity with SEQ ID NO: 33. In particularembodiments, a MpcII-3-related protein has at least 70%, 80%, 90%, 95%or more amino acid identity with SEQ ID NO: 33.

The present invention further provides a method of isolating one or morehoming molecules that selectively homes to heart vasculature bycontacting bc10, or a fragment thereof, with a library of moleculesunder conditions suitable for specific binding of a molecule to bc10;assaying for specific binding; and separating one or more bc10-bindingmolecules from the library, thereby isolating one or more homingmolecules that selectively homes to heart vasculature and specificallybinds bc10. Cells that express bc10 on the cell surface as well aspurified bc10, or a fragment of bc10, are useful in the screeningmethods of the invention. As non-limiting examples, native, recombinant,human, murine (SEQ ID NO: 35) as well as variants and other specieshomologs of bc10, and fragments thereof, including, without limitation,SEQ ID NO: 12-binding fragments or SEQ ID NO: 13-binding fragments,whether purified or expressed on the surface of a cell, can be useful inthe screening methods of the invention. The methods of the invention areuseful for screening any of a variety of libraries as discussed above.

The term “bc10,” as used herein, means a polypeptide which isselectively expressed in heart vasculature and which has at least 60%amino acid identity with SEQ ID NO: 35. In particular embodiments, bc10has at least 70%, 80%, 90%, 95% or more amino acid identity with SEQ IDNO: 35.

The present invention further provides a variety of isolated peptidesand peptidomimetics, which can be useful, for example, in constructingthe conjugates of the invention or, where the peptide itself hasbiological activity, in unconjugated form as a therapeutic for treatingany cardiovascular disease or cardiomyopathy as described above. Otherhoming peptides with biological activity are known in the art asdescribed, for example, in Ruoslahti, Nat. Rev. Cancer 2:83-90 (2002),and Laakkonen et al., Proc. Natl. Acad. Sci. USA 101:9381-9386 (2004).As one example, the present invention provides an isolated peptide orpeptidomimetic which has a length of less than 60 residues and includesthe amino acid sequence CRPPR (SEQ ID NO: 1) or a peptidomimeticthereof. In one embodiment, the invention provides an isolated peptidewhich has a length of less than 60 residues and includes the amino acidsequence CRPPR (SEQ ID NO: 1). An isolated peptide or peptidomimetic ofthe invention which includes the amino acid sequence CRPPR (SEQ IDNO: 1) can have, for example, a length of less than 40 residues or alength of less than 20 residues.

The invention further provides an isolated peptide or peptidomimeticwhich includes the amino acid sequence GRKSKTV (SEQ ID NO: 14) or apeptidomimetic thereof. In one embodiment, the invention provides anisolated peptide which includes the amino acid sequence GRKSKTV (SEQ IDNO: 14). An isolated peptide or peptidomimetic of the invention can be,for example, conformationally constrained or can include the amino acidsequence CGRKSKTVC (SEQ ID NO: 2). In further embodiments, an isolatedpeptide or peptidomimetic which includes the amino acid sequence GRKSKTV(SEQ ID NO: 14) or a peptidomimetic thereof has a length of less than100 residues, a length of less than 60 residues or a length of less than20 residues. In yet a further embodiment, the invention provides anisolated peptide or peptidomimetic which includes the amino acidsequence CXGRKSKTVZC (SEQ ID NO: 15) or a peptidomimetic thereof, whereX=0 to 20 independently selected residues and Z=0 to 20 independentlyselected residues.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 150 residues and includesthe amino acid sequence CARPAR (SEQ ID NO: 5) or a peptidomimeticthereof. In one embodiment, the invention provides an isolated peptidewhich has a length of less than 150 residues and includes the amino acidsequence CARPAR (SEQ ID NO: 5). The isolated peptides andpeptidomimetics of the invention which includes the amino acid sequenceCARPAR (SEQ ID NO: 5) can have, without limitation, a length of lessthan 100 residues, 60 residues or 20 residues.

Also provided herein is an isolated peptide or peptidomimetic which hasa length of less than 50 residues and includes the amino acid sequenceCPKRPR (SEQ ID NO: 6) or a peptidomimetic thereof. In one embodiment,the invention provides an isolated peptide which has a length of lessthan 50 residues and includes the amino acid sequence CPKRPR (SEQ ID NO:6). The isolated peptides and peptidomimetics of the invention whichinclude the amino acid sequence SEQ ID NO: 6 can have a variety oflengths including, but not limited to, a length of less than 40 residuesor a length of less than 20 residues.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence CKRAVR (SEQ ID NO: 7) or a peptidomimeticthereof. In one embodiment, the invention provides an isolated peptidewhich has a length of less than 400 residues and includes the amino acidsequence CKRAVR (SEQ ID NO: 7). A peptide or peptidomimetic of theinvention including the amino acid sequence CKRAVR (SEQ ID NO: 7) canhave, without limitation, a length of less than 100 residues, a lengthof less than 60 residues or a length of less than 20 residues.

Further provided by the invention is an isolated peptide orpeptidomimetic which includes the amino acid sequence RNSWKPN (SEQ IDNO: 16) or a peptidomimetic thereof. In one embodiment, the inventionprovides an isolated peptide which includes the amino acid sequenceRNSWKPN (SEQ ID NO: 16). As non-limiting examples, an isolated peptideor peptidomimetic of the invention can be conformationally constrainedor can have a length of less than 100 residues, a length of less than 60residues or a length of less than 20 residues. In one embodiment, theinvention provides an isolated peptide or peptidomimetic which includesthe amino acid sequence CXRNSWKPNZC (SEQ ID NO: 17) or a peptidomimeticthereof, where X=0 to 20 independently selected residues and Z=0 to 20independently selected residues.

Further provided herein is an isolated peptide or peptidomimetic whichincludes the amino acid sequence RGSSS (SEQ ID NO: 9) or apeptidomimetic thereof. In one embodiment, the invention provides anisolated peptide containing the amino acid sequence RGSSS (SEQ ID NO:9). In further embodiments, an isolated peptide or peptidomimetic of theinvention containing the amino acid sequence SEQ ID NO: 9 has a lengthof less than 100 residues, a length of less than 60 residues or a lengthof less than 20 residues.

The present invention also provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence RSTRANP (SEQ ID NO: 18) or a peptidomimeticthereof. The invention provides, for example, an isolated peptide whichhas a length of less than 400 residues and includes the amino acidsequence RSTRANP (SEQ ID NO: 18). As a non-limiting example, an isolatedpeptide or peptidomimetic of the invention can be conformationallyconstrained. As a further non-limiting example, an isolated peptide orpeptidomimetic of the invention can include the amino acid sequenceCRSTRANPC (SEQ ID NO: 11). An isolated peptide or peptidomimetic of theinvention further can have any of a variety of lengths. As non-limitingexamples, an isolated peptide or peptidomimetic of the invention canhave a length of less than 100 residues, less than 60 residues or lessthan 20 residues. In one embodiment, an isolated peptide orpeptidomimetic of the invention includes the amino acid sequenceCXRSTRANPZC (SEQ ID NO: 19) or a peptidomimetic thereof, where X=0 to 20independently selected residues and Z=0 to 20 independently selectedresidues.

The present invention additionally provides an isolated peptide orpeptidomimetic which has a length of less than 400 residues and includesthe amino acid sequence PKTRRVP (SEQ ID NO: 20) or a peptidomimeticthereof. In one embodiment, the invention provides a peptide which has alength of 400 residues and includes the amino acid sequence PKTRRVP (SEQID NO: 20). An isolated peptide or peptidomimetic of the invention canbe, for example, conformationally constrained, or can include the aminoacid sequence CPKTRRVPC (SEQ ID NO: 12). In particular embodiments, anisolated peptide or peptidomimetic of the invention containing the aminoacid sequence SEQ ID NO: 20 has a length of less than 100 residues, lessthan 60 residues or less than 20 residues. In a further embodiment, anisolated peptide or peptidomimetic includes the amino acid sequenceCXPKTRRVPZC (SEQ ID NO: 21) or a peptidomimetic thereof, where X=0 to 20independently selected residues and Z=0 to 20 independently selectedresidues.

Further provided herein is an isolated peptide or peptidomimetic whichhas a length of less than 400 residues and includes the amino acidsequence SGMARTK (SEQ ID NO: 22) or a peptidomimetic thereof. In oneembodiment, the invention provides a peptide which has a length of lessthan 400 residues and includes the amino acid sequence SGMARTK (SEQ IDNO: 22). In another embodiment, an isolated peptide or peptidomimetic ofthe invention is conformationally constrained. In a further embodiment,an isolated peptide or peptidomimetic of the invention includes theamino acid sequence CSGMARTKC (SEQ ID NO: 13). An isolated peptide orpeptidomimetic of the invention further can have any of a variety oflengths. As non-limiting examples, an isolated peptide or peptidomimeticof the invention having the amino acid sequence SEQ ID NO: 22 can have alength of less than 100 residues, 60 residues or 20 residues. In oneembodiment, the invention provides an isolated peptide or peptidomimeticwhich includes the amino acid sequence CXSGMARTKZC (SEQ ID NO: 23) or apeptidomimetic thereof, where X=0 to 20 independently selected residuesand Z=0 to 20 independently selected residues.

The peptides and peptidomimetics of the invention are provided inisolated form. As used herein in reference to a peptide orpeptidomimetic of the invention, the term “isolated” means a peptide orpeptidomimetic that is in a form that is relatively free from materialsuch as contaminating polypeptides, lipids, nucleic acids and othercellular material that normally is associated with the peptide orpeptidomimetic in a cell or that is associated with the peptide orpeptidomimetic in a library or in a crude preparation.

Thus, the invention provides peptides and peptidomimetics includingconformationally constrained, bifunctional and multivalent peptides andpeptidomimetics as disclosed below. As used herein, the term “peptide”is used broadly to mean peptides, proteins, fragments of proteins andthe like. The term “peptidomimetic,” as used herein, means apeptide-like molecule that has the activity of the peptide upon which itis structurally based. Such peptidomimetics include chemically modifiedpeptides, peptide-like molecules containing non-naturally occurringamino acids, and peptoids, and have an activity such as the selectivehoming activity of the peptide upon which the peptidomimetic is derived(see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in“Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E.Wolff; John Wiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art and are encompassedwithin the invention including, for example, peptide-like moleculeswhich contain a constrained amino acid, a non-peptide component thatmimics peptide secondary structure, or an amide bond isostere. Apeptidomimetic that contains a constrained, non-naturally occurringamino acid can include, for example, an α-methylated amino acid;α,α-dialkylglycine or α-aminocycloalkane carboxylic acid; anN^(α)—C^(α)cyclized amino acid; an N^(α)-methylated amino acid; a β- orγ-amino cycloalkane carboxylic acid; an α,β-unsaturated amino acid; aβ,β-dimethyl or β-methyl amino acid; a β-substituted-2,3-methano aminoacid; an N—C^(δ) or C^(δ)—C^(δ) cyclized amino acid; a substitutedproline or another amino acid mimetic. A peptidomimetic which mimicspeptide secondary structure can contain, for example, a nonpeptidicβ-turn mimic; γ-turn mimic; mimic of β-sheet structure; or mimic ofhelical structure, each of which is well known in the art. Apeptidomimetic also can be a peptide-like molecule which contains, forexample, an amide bond isostere such as a retro-inverso modification;reduced amide bond; methylenethioether or methylene-sulfoxide bond;methylene ether bond; ethylene bond; thioamide bond; trans-olefin orfluoroolefin bond; 1,5-disubstituted tetrazole ring; ketomethylene orfluoroketomethylene bond or another amide isostere. One skilled in theart understands that these and other peptidomimetics are encompassedwithin the meaning of the term “peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptide of theinvention, as well as potential geometrical and chemical complementarityto a cognate receptor. Where no crystal structure of a peptide of theinvention is available, a structure can be generated using, for example,the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251(1989)). Another database, the Available Chemicals Directory (MolecularDesign Limited, Informations Systems; San Leandro Calif.), containsabout 100,000 compounds that are commercially available and also can besearched to identify potential peptidomimetics of a peptide of theinvention, for example, with activity in selectively homing to heartvasculature.

The peptides and peptidomimetics of the invention, including theconformationally constrained, bifunctional and multivalent peptides andpeptidomimetics discussed below, can have a variety of lengths. Apeptide or peptidomimetic of the invention can have, for example, arelatively short length of less than six, seven, eight, nine, ten, 12,15, 20, 25, 30, 35, 40, 45, 50, 60, 70 or 80 residues. A peptide orpeptidomimetic of the invention also can be useful in the context of asignificantly longer sequence as described further below. As usedherein, the term “residue” refers to amino acids or analogs thereof. Itis understood that a peptide containing, for example, the amino acidsequence SEQ ID NO: 1 includes the specified amino acids as a contiguoussequence not separated by other amino acids.

An isolated peptide or peptidomimetic of the invention can be, withoutlimitation, cyclic or otherwise conformationally constrained. As usedherein in reference to a molecule, the term “conformationallyconstrained” means a molecule, such as a peptide or peptidomimetic, inwhich the three-dimensional structure is maintained substantially in onespatial arrangement over time. Conformationally constrained moleculescan have improved properties such as increased affinity, metabolicstability, membrane permeability or solubility. Methods ofconformational constraint are well known in the art and include, withoutlimitation, cyclization.

As used herein in reference to a peptide or peptidomimetic, the termcyclic refers to a structure including an intramolecular bond betweentwo non-adjacent amino acids or amino acid analogs. The cyclization canbe effected through a covalent or non-covalent bond. Intramolecularbonds include, but are not limited to, backbone to backbone, side-chainto backbone, and side-chain to side-chain bonds. Methods of cyclizationinclude, without limitation, formation of a disulfide bond between theside-chains of non-adjacent amino acids or amino acid analogs; formationof a lactam bond, for example, between a side-chain group of one aminoacid or analog thereof to the N-terminal amine of the amino-terminalresidue; and formation of lysinonorleucine and dityrosine bonds.

The present invention also provides an isolated peptide orpeptidomimetic containing an amino acid sequence which is a conservativevariant, for example, of the sequence CRPPR (SEQ ID NO: 1), CGRKSKTVC(SEQ ID NO: 2), CARPAR (SEQ ID NO: 5), CPKRPR (SEQ ID NO: 6), CKRAVR(SEQ ID NO: 7), CRNSWKPNC (SEQ ID NO: 8), RGSSS (SEQ ID NO: 9),CRSTRANPC (SEQ ID NO: 11), CPKTRRVPC (SEQ ID NO: 12) or CSGMARTKC (SEQID NO: 13). In particular embodiments, the invention provides anisolated peptide or peptidomimetic containing an amino acid sequencewhich is a conservative variant of SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 11,12 or 13, in which exactly one amino acid is conservatively substituted.In further embodiments, the invention provides an isolated peptide orpeptidomimetic containing an amino acid sequence which is a conservativevariant of SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 11, 12 or 13, in whichexactly two amino acids are conservatively substituted. In furtherembodiments, the invention provides an isolated peptide orpeptidomimetic containing an amino acid sequence which is a conservativevariant of SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 11, 12 or 13, in whichexactly three, four or five amino acids are conservatively substituted.

As used herein, a “conservative variant” is an amino acid sequence inwhich a first amino acid is replaced by a second amino acid or aminoacid analog having at least one similar biochemical property, which canbe, for example, similar size, charge, hydrophobicity orhydrogen-bonding capacity. For example, a first hydrophobic amino acidcan be conservatively substituted with a second (non-identical)hydrophobic amino acid such as alanine, valine, leucine, or isoleucine,or an analog thereof. Similarly, a first basic amino acid can beconservatively substituted with a second basic amino acid such asarginine or lysine, or an analog thereof. In the same way, a firstacidic amino acid can be conservatively substituted with a second acidicamino acid such as aspartic acid or glutamic acid, or an analog thereof,or an aromatic amino acid such as phenylalanine can be conservativelysubstituted with a second aromatic amino acid or amino acid analog, forexample, tyrosine.

As disclosed herein, a peptide or peptidomimetic that selectively homesto heart vasculature can maintain homing activity in the context of asignificantly longer sequence. For example, the 5-mer CRPPR (SEQ IDNO: 1) maintained the ability to home when fused to a phage coatprotein, confirming that a peptide of the invention can have selectivehoming activity when embedded in a larger protein sequence. Thus, theinvention provides chimeric proteins which contain a peptide orpeptidomimetic of the invention fused to a heterologous protein. Incertain embodiments, the invention provides a chimeric proteincontaining a homing peptide or peptidomimetic that selectively homes toheart vasculature and that specifically binds HLP/CRIP2, receptor clone9, Sigirr/TIR8, MpcII-3-related protein, or bc-10 fused to aheterologous protein. Heterologous proteins useful in the inventioninclude, without limitation, those having therapeutic activity as wellantibodies or antigen-binding fragments thereof. In further embodiments,the invention provides a chimeric protein in which a peptide orpeptidomimetic containing the amino acid sequence SEQ ID NO: 1, 2, 5, 6,7, 8, 9, 11, 12 or 13, or a conservative variant or peptidomimetic ofone of these sequences, is fused to a heterologous protein. The term“heterologous,” as used herein in reference to a protein fused to apeptide or peptidomimetic of the invention, means a protein derived froma source other than the gene encoding the fused peptide or from whichthe fused peptidomimetic is derived. A chimeric protein of the inventioncan have a variety of lengths including, but not limited to, up to 100,200, 300, 400, 500, 800, 1000 or 2000 residues.

Further provided herein are bifunctional peptides which contain a homingpeptide that selectively homes heart vasculature and that specificallybinds HLP/CRIP2 (SEQ ID NO: 25), receptor clone 9 (SEQ ID NO: 27),Sigirr/TIR8 (SEQ ID NO: 29), the MpcII-3 related protein SEQ ID NO: 33,or bc-10 (SEQ ID NO: 35), fused to a second peptide having a separatefunction. Such bifunctional peptides have at least two functionsconferred by different portions of the peptide and can, for example,display pro-apoptotic activity in addition to selective homing activity.

The present invention further provides an isolated multivalent peptideor peptidomimetic that includes at least two motifs each independentlybinding HLP/CRIP2 (SEQ ID NO: 25). Such a multivalent peptide orpeptidomimetic can have, for example, at least two motifs eachindependently containing the amino acid sequence SEQ ID NO: 1 or SEQ IDNO: 2, or a conservative variant or peptidomimetic thereof. Themultivalent peptide or peptidomimetic can have, for example, at leastthree, at least five or at least ten of such motifs, each independentlycontaining the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or aconservative variant or peptidomimetic thereof. In particularembodiments, the multivalent peptide or peptidomimetic has two, three,four, five, six, seven, eight, nine, ten, fifteen or twenty identical ornon-identical motifs of the amino acid sequence of SEQ ID NO: 1 or SEQID NO: 2, or a conservative variant or peptidomimetic thereof. Inanother embodiment, the multivalent peptide or peptidomimetic containsidentical motifs, which consist of the amino acid sequence SEQ ID NO: 1,or a conservative variant or peptidomimetic of this sequence. In yetanother embodiment, the multivalent peptide or peptidomimetic containsidentical motifs, which consist of the amino acid sequence SEQ ID NO: 2,or a conservative variant or peptidomimetic of this sequence. In stillfurther embodiments, the multivalent peptide or peptidomimetic containscontiguous HLP/CRIP2-binding motifs, which can be identical ornon-identical.

Also provided herein is an isolated multivalent peptide orpeptidomimetic that includes at least two motifs each independentlybinding receptor clone 9 (SEQ ID NO: 27). The multivalent peptides orpeptidomimetics of the invention can have, for example, at least twomotifs each independently containing the amino acid sequence SEQ ID NO:5 or 6, or a conservative variant or peptidomimetic of these sequences.It is understood that a multivalent peptide or peptidomimetic of theinvention can include, for example, at least three, at least five or atleast ten of such motifs, each independently containing the amino acidsequence of SEQ ID NO: 5 or 6, or a conservative variant orpeptidomimetic thereof. In particular embodiments, the multivalentpeptide or peptidomimetic has two, three, four, five, six, seven, eight,nine, ten, fifteen or twenty identical or non-identical motifs of theamino acid sequence of SEQ ID NO: 5 or 6, or a conservative variant orpeptidomimetic thereof. A multivalent peptide or peptidomimetic of theinvention can include, for example, identical motifs consisting of theamino acid sequence of SEQ ID NO: 5 or 6, or a conservative variant orpeptidomimetic of such a sequence. In further embodiments, themultivalent peptide or peptidomimetic contains contiguous receptor clone9 (SEQ ID NO: 27)-binding motifs, which are identical or non-identical.

The present invention further provides an isolated multivalent peptideor peptidomimetic that includes at least two motifs each independentlybinding Sigirr/TIR8 (SEQ ID NO: 29). The multivalent peptides orpeptidomimetics of the invention can have, for example, at least twomotifs each independently containing the amino acid sequence SEQ ID NO:7, 8 or 9, or a conservative variant or peptidomimetic of thesesequences. It is understood that a multivalent peptide or peptidomimeticof the invention can include, for example, at least three, at least fiveor at least ten of such motifs, each independently containing the aminoacid sequence of SEQ ID NO: 7, 8 or 9, or a conservative variant orpeptidomimetic thereof. In particular embodiments, the multivalentpeptide or peptidomimetic has two, three, four, five, six, seven, eight,nine, ten, fifteen or twenty identical or non-identical motifs of theamino acid sequence of SEQ ID NO: 7, 8 or 9, or a conservative variantor peptidomimetic thereof. A multivalent peptide or peptidomimetic ofthe invention can include, for example, identical motifs consisting ofthe amino acid sequence of SEQ ID NO: 7, 8 or 9, or a conservativevariant or peptidomimetic of such a sequence. In further embodiments,the multivalent peptide or peptidomimetic contains contiguousSigirr/TIR8 (SEQ ID NO: 29)-binding motifs, which are identical ornon-identical.

Further provided herein is an isolated multivalent peptide orpeptidomimetic that includes at least two motifs each independentlybinding SEQ ID NO: 33. The multivalent peptides or peptidomimetics ofthe invention can have, for example, at least two motifs eachindependently containing the amino acid sequence CRSTRANPC (SEQ ID NO:11), or a conservative variant or peptidomimetic of these sequences. Asnon-limiting examples, a multivalent peptide or peptidomimetic of theinvention can include at least three, at least five or at least ten ofsuch motifs, each independently containing the amino acid sequence ofCRSTRANPC (SEQ ID NO: 11), or a conservative variant or peptidomimeticthereof. As further non-limiting examples, a multivalent peptide orpeptidomimetic can have two, three, four, five, six, seven, eight, nine,ten, fifteen or twenty identical or non-identical motifs of the aminoacid sequence of CRSTRANPC (SEQ ID NO: 11), or a conservative variant orpeptidomimetic thereof. A multivalent peptide or peptidomimetic of theinvention also can include, for example, identical motifs consisting ofthe amino acid sequence of SEQ ID NO: 11, or a conservative variant orpeptidomimetic of such a sequence. It is understood that a multivalentpeptide or peptidomimetic can include contiguous or non-contiguous SEQID NO: 33-binding motifs, which can be identical or non-identical.

Also provided by the present invention is an isolated multivalentpeptide or peptidomimetic that includes at least two motifs which eachindependently bind bc10 (SEQ ID NO: 35). The (SEQ ID NO: 35)-bindingmotifs can be contiguous or non-contiguous, and further can be identicalor non-identical. A multivalent peptide or peptidomimetic of theinvention can have, for example, at least two motifs each independentlycontaining the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, ora conservative variant or peptidomimetic of these sequences. Asnon-limiting examples, a multivalent peptide or peptidomimetic of theinvention can include at least three, at least five or at least ten ofsuch motifs, each independently containing the amino acid sequence ofSEQ ID NO: 12 or SEQ ID NO; 13, or a conservative variant orpeptidomimetic thereof. As further non-limiting examples, a multivalentpeptide or peptidomimetic can have two, three, four, five, six, seven,eight, nine, ten, fifteen or twenty identical or non-identical motifs ofthe amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, or aconservative variant or peptidomimetic thereof. In one embodiment, theinvention provides a multivalent peptide or peptidomimetic of theinvention which includes identical motifs consisting of the amino acidsequence of SEQ ID NO: 12 or SEQ ID NO: 13, or a conservative variant orpeptidomimetic of SEQ ID NO: 12 or SEQ ID NO: 13.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Identification of Peptides that Selectively Home to HeartVasculature and Cognate Receptors For the Homing Peptides

This example describes the use of ex vivo and in vivo phage selectionfor identification of peptides that selectively home to heartvasculature, combined with bacterial two-hybridization assays foridentification of cognate receptors for the homing peptides.

Ex vivo phage selection for enrichment of cardiac endothelial cells wasperformed on cell suspensions obtained from murine heart tissue usinganti-CD31 magnetic beads for isolation of vascular endothelial cells. A230-fold enrichment in ex vivo binding to heart cells as compared tonon-recombinant phage was observed following three rounds of ex vivoselection (FIG. 1B). Subsequent in vivo selection for homing to theheart resulted in a phage pool that homed to the heart in vivo with anincrease of nearly 200-fold relative to the homing of non-recombinantphage. Furthermore, the ex vivo/in vivo selected phage preferentiallylocalized to the heart as compared to other tissues. As shown in FIG.1C, the enrichment in heart was 20 to 50-fold as compared to phageaccumulation in brain, kidney, skin and skeletal muscle.

Isolation of putative receptors for the heart-homing peptides wasperformed as follows. Peptide-encoding DNA inserts of the heart-homingphage were amplified by polymerase chain reaction (PCR); introduced intoa “bait vector” of a bacterial two-hybrid system; and co-transformedinto bacteria with a target vector encoding a heart cDNA library. A highstringency screen revealed colonies indicative of a bait-targetinteraction at a frequency comparable to the positive control, whilecolony growth was not observed in the absence of either of the twovectors (FIG. 1D). Furthermore, a control for leakiness of the systemeliminated 75 out of 100 colonies as likely false positives (FIG. 1E).

In-frame sequences from the 25 remaining candidate receptor cDNAs wereanalyzed using the NCBI non-redundant database. Of these 25 clones, 19were proteins abundantly expressed in cardiac muscle, such as myosin,myoglobin, ferritin and oxoglutarate dehydrogenase. The remaining sixclones, which represent membrane or cell surface proteins as summarizedin Table 1, are putative receptors for heart homing molecules. Theseresults indicate that the combination of ex vivo/in vivo phage displayand bacterial two-hybrid analysis can rapidly identify heart-homingpeptides and their cognate receptors.

Ex vivo and in vivo phage selections were performed as follows. AnNNK-encoded CX₇C library displayed on T7Select415-1 phage (Novagen; SanDiego, Calif.) was generated as described in Laakkonen et al., Nat. Med.8:751-755 (2002). Phage selections and validations were performed asdescribed in Hoffman et al., “In vivo and ex vivo selections usingphage-displayed libraries,” in Clarkson and Lowman (Eds.) Phage Display:A Practical Approach Oxford, U.K.: Oxford University Press (2004)).Following three rounds of ex vivo selection on heart cells, three roundsof selection were performed in vivo. For ex vivo selections, cellsuspensions were prepared from murine heart tissue using collagenase IAat 1 mg/ml (Sigma; St. Louis, Mo.) to disperse the tissue. Cellsuspensions were incubated overnight at 4° C. with 5×10¹⁰ plaque formingunits (pfu) of a CX₇C library and subsequently washed to remove unboundphage. Magnetic beads (M450; Dynal; Oslo, Norway) and anti-mouse CD31(PharMingen; San Diego, Calif.) were used to isolate vascularendothelial cells according to the manufacturer's instructions. Phagebound to the CD31-positive cell population were rescued and amplified inE. coli as described in Hoffman et al., supra, 2004.

In vivo panning was performed as follows. The ex vivo-selected phagepool (5×10⁹ p.f.u.) was intravenously injected into mice through thetail vein and allowed to circulate for ten minutes before mice werecarefully perfused through the left ventricle with DMEM in order toremove unbound intravascular phage. The heart and control tissues(brain, kidney, skin and muscle) were excised, and phage recovered asdescribed in Hoffman et al., supra, 2004. Phage recovered from the heartwere reinjected into mice, and the cycle was repeated for a total ofthree rounds. Non-recombinant phage were injected into a separate mouseas a control in each experiment.

For bacterial two-hybridization experiments, a library of bait plasmidswas constructed by PCR amplification of peptide-encoding DNA insertsfrom the heart-homing phage using primers 5′-TCAGGTGTGATGCTCGG-3′ (SEQID NO: 36; forward primer) and 5′-GAGTAACTAGTTAACC-3′ (SEQ ID NO: 31;reverse primer). PCR products were digested with EcoRI and XhoI, andsubcloned into the bait pBT plasmid (Stratagene; La Jolla, Calif.) togenerate a recombinant library of baits designated “pBT-hp.” pBT-hpplasmids were transformed into XL1-Blue MRF’ Kan competent cells, and 2μl or 5 μl of cells plated on 100 mm LB-chloramphenicol plates todetermine the total number of primary transformants. The remainingtransformants were pooled and plated on 150 mm LB-chloramphenicolplates. Sequencing was performed on 20 clones, which were found to haveproper peptide-encoding inserts. The plates were then scraped, andbacteria harvested. After plating 2 μl and 5 μl of the harvested libraryin order to determine the size of the amplified library, the pool ofpBT-hp plasmids was purified from the pooled bacteria using acommercially available maxiprep DNA column. Purified DNA was used totransform BACTERIOMATCH® Two-Hybrid System reporter strain competentcells according to the manufacturer's instructions (Stratagene; LaJolla, Calif.).

Heart cDNA library plasmids were plate purified by diluting 2 μl ofcells containing mouse heart plasmid cDNA library (Stratagene; #982303)into 25 ml SOC medium, and then spreading the cells onto twenty-five25-cm×25-cm LB-tetracycline plates. Plates were incubated at 30° C. for24 hours before harvesting of approximately 5×10⁶ individual clones andsubsequent plasmid purification.

Bacterial two-hybridization experiments were performed with theBACTERIOMATCH® Two-Hybrid System (Stratagene) according to themanufacturer's instructions. Briefly, pBT-hp and heart cDNA libraryplasmids were co-transformed using 50 ng each plasmid. Aliquots (100 μl)of co-transformants were plated on selective LB plates (500 μg/mlcarbenicillin) and incubated at 37° C. for 20 hours before scoring theplates for growth. An interaction between a pair of hybrid proteins wasindicated by growth on selective plates, as observed for the positivecontrols, Gal4 and Gal11P hybrid protein. Individual positive coloniesfrom carbenicillin plates were subsequently streaked onto X-gal platesfor a secondary screen. After incubation of the cells for 12 to 14hours, cells were selected that showed a dark blue color with X-galplates, and were able to grow in the presence of LB-chloramphenicol orLB-tetracycline (markers of the target and bait plasmids) overnight at30° C. Plasmid DNA was purified, and the reporter strain re-transformedwith the putative bait and target plasmids. Clones which grewreproducibly on selective plates were verified as positive. Bait andtarget inserts were amplified by PCR prior to sequence analysis.Sequences from target vectors were used as queries in a BLASTN search ofthe nonredundant mouse genome.

Example II Receptor-Peptide Pairs and Peptide Heart-Homing Activity

This example describes the membrane or cell surface expressed proteinswhich were identified as putative receptors for heart-homing peptides bybacterial two-hybrid analysis with a peptide pool displaying hearthoming activity.

Receptor Clone 5

As summarized in Table 1, receptor clone 5 represents thecarboxy-terminal 92 amino acids of heart LIM-protein (HLP), alsodesignated cysteine-rich protein 2 (CRIP2) and ESP 1. HLP, which isexpressed in the vascular heart (Yu et al., Mech. Dev. 116:187-192(2002)) is a LIM domain-containing protein having homology to Crp-1(Karim et al., Genomics 31:167-176 (1996); and van Ham et al., GenesCells 8:631-644 (2003)). Of the five positive colonies from thetwo-hybrid analysis with the HLP clone, three cognate peptides wereidentified: CRPPR (SEQ ID NO: 1) was present twice, CGRKSKTVC (SEQ IDNO: 2) once and CGNQVDSRC (SEQ ID NO: 3) twice. After cloning thesepeptides into the T7Select 415-1 display vector, they were assayed fortheir ability to home to heart vasculature following intravenousinjection. Relative to non-recombinant phage, the CRPPR (SEQ ID NO:1)-displaying phage homed with more than 300 fold selectivity to theheart (FIG. 2A). As summarized in Table 1, CGRKSKTVC (SEQ ID NO:2)-phage displayed about 50-fold selectivity in heart-homing while nosignificant selective homing was observed for phage displaying CGNQVDSRC(SEQ ID NO: 3).

Receptor Clone 9

Receptor clone 9 was also identified as a membrane protein which bindsto one or more heart-homing peptides. As indicated in Table 1, receptorclone 9 is an unannotated RIKEN EST. Of the five colonies identifiedthrough two-hybrid analysis with receptor clone 9, three peptides,CPSELLLP (SEQ ID NO: 4), CARPAR (SEQ ID NO: 5) and CPKRPR (SEQ ID NO:6), were identified. The latter two peptides exhibited selective homingto the heart while the first did not (Table I and FIG. 2B).

Receptor Clone 15

Receptor clone 15 is a single Ig IL-1 receptor-related proteindesignated Sigirr or TIR8 (Thomassen et al., Cytokine 11:389-399(1999)). The portion of the protein represented in clone 15 containedthe transmembrane domain of Sigirr as well as an amino-terminalextracellular portion of about 190 amino acids. Sigirr is known to beexpressed in the epithelia of the kidneys, lungs, gut and other tissues(Thomassen et al., supra, 1999; and Polentarutti et al., Eur. CytokineNetw. 14:211-218 (2003)), although heart or endothelial cell expressionhas not been reported. As summarized in Table 1, three peptides wereidentified as binding Sigirr through two-hybrid analysis. Thesepeptides, CKRAVR (SEQ ID NO: 7), CRNSWKPNC (SEQ ID NO: 8), and RGSSS(SEQ ID NO: 9), showed 20 to 30-fold heart homing selectivity (see Table1 and FIG. 2C).

Receptor Clone 27

Receptor clone 27 is a hypothetical protein annotated as aglutamine-rich region containing protein identified from an olfactorycDNA library. The five bait clones analyzed all encoded the samepeptide, CLIDLHVMC (SEQ ID NO: 10), which showed no specific homing tothe heart (Table 1 and FIG. 2D).

Receptor Clone 36

Receptor clone 36 contains the entire coding sequence of an unnamedprotein product from the RIKEN Fantom set. This receptor is putativelysimilar to integral membrane protein CII-3 (MpcII-3), a mitochondrialmembrane protein that is part of the succinate dehydrogenase complex.Each of the five sequenced colonies identified though binding-activityto clone 36 encoded the same peptide, CRSTRANPC (SEQ ID NO: 5), whichdisplayed about 20-fold heart homing selectivity (Table 1 and FIG. 2E).

Receptor Clone 46

Receptor clone 46 is a portion of the mouse homolog of the human bladdercancer-associated protein 10 (bc10), a small membrane protein which isdown regulated as cancer develops from pre-malignant lesions in thebladder (Gromova et al., Int. J. Cancer 98:539-546 (2002)). The fragmentisolated as receptor clone 46 contains the carboxy-terminal 32 aminoacids of the predicted extracellular domain of bc10. Two-hybrid analysisidentified two peptides which bound receptor clone 46: CPKTRRVPC (SEQ IDNO: 12) and CSGMARTKC (SEQ ID NO: 13), which demonstrated about 60- and10-fold heart homing selectivity, respectively (Table 1 and FIG. 2F).

In sum, these results indicate that bacterial two-hybrid analysis,combined with in vivo panning, can be useful for identifying bothorgan-homing peptides and their cognate receptors.

Individual phage clones were reconstructed as follows. Oligonucleotidesthat encoded peptides from selected bait plasmids were synthesized,phosphorylated with T4 polynucleotide kinase (PNK; NEB) at 37° C. forone hour, and annealed at a concentration of 0.08 μmol/μl. The annealedinserts were diluted to 0.04 pmol/μl and ligated into T7Select 415-1arms (Novagen) with T4 ligase overnight at 16° C. The next day, intactrecombinant phage were prepared by mixing the ligation reaction withpackaging extracts as described by the manufacturer. Inserts wereconfirmed by sequencing, and individual clones were amplified for exvivo and in vivo testing as described in Example I above.

Example III Phage-Displayed Heart Homing Peptides Localize to HeartVasculature

This example demonstrates that the disclosed peptides which selectivelyhome to heart vasculature specifically bind receptors which areexpressed in vivo by heart vasculature.

A. Phage-Displayed Heart Homing Peptides Co-Localize with a VascularMarker

Further characterization was performed on the five most efficient homingpeptides, as indicated by the fold-selectivity summarized in Table 1,and their putative receptors. These homing peptides were analyzed forthe ability to selectively bind heart endothelia in vivo and forspecific binding to their putative receptors, which also were analyzedfor expression in heart endothelia. Each of the phage-displaying aparticular homing peptide was individually injected intravenously intomice with fluorescein-conjugated tomato lectin, a blood vessel marker.Localization of phage with anti-T7 antibody showed that phage displayingpeptide CRPPR (SEQ ID NO: 1), CARPAR (SEQ ID NO: 5), CKRAVR (SEQ ID NO:7), CRSTRANPC (SEQ ID NO: 11) or CPKTRRVPC (SEQ ID NO: 12) were presentin heart endothelia (FIGS. 3A to C, E and F). The CLIDLHVMC (SEQ ID NO:10)-displaying phage served as a negative control (FIG. 3D), and wasalso absent from the heart by phage immunostaining (FIG. 3D). None ofthe six phage were detected in tissues other than heart under the sameconditions.

These results demonstrate that phage displaying peptide CRPPR (SEQ IDNO: 1), CARPAR (SEQ ID NO: 5), CKRAVR (SEQ ID NO: 7), CRSTRANPC (SEQ IDNO: 11) or CPKTRRVPC (SEQ ID NO: 12) localize to heart vasculature invivo.

Immunohistochemistry was performed as follows. Rat monoclonal anti-mouseCD31 (1:100) was obtained from BD Pharmingen (San Jose, Calif.), andrabbit polyclonal anti-T7 phage (1:500), and chicken anti-mouse CRIP2IgY (1:100) were obtained from GenWay (San Diego, Calif.). Secondaryantibodies, AlexaFluor-488 goat anti-rat IgG (1:1000) and AlexaFluor-594goat anti-rat or rabbit IgG (1:1000) were obtained from Molecular Probes(Eugene, Oreg.); the secondary antibody GAYFC-AlexaFluor 594 goatanti-IgY Fc (1:250) was obtained from GenWay. Frozen sections werepreincubated with blocking buffer (5% normal goat serum and 0.5% BSA in1×PBS) for one hour, washed three times with PBS, and incubated with theprimary antibody of interest overnight at 4° C. After the overnightincubation, secondary antibodies were added and incubated for one hourat room temperature. Slides were washed three times with PBS and mountedin Vectashield Mounting Medium with DAPI (Vector Laboratories;Burlingame, Calif.). Blood vessels were additionally visualized byintravenous injection with Lycopersicon esculentum (tomato) lectinconjugated to fluorescein (100 μg in 200 μl of PBS) purchased fromVector Laboratories.

B. Homing Peptides Binds to Cells Transfected with their PutativeCognate Receptors

Full-length cDNAs were constructed for four of the putative heart homingreceptors, HLP/CRIP2, Sigirr/TIR8, and MpcII-3-related protein and bc10,and expressed in 293T cells. As shown in FIG. 4, phage displaying eachof the four cognate peptides bound to cells transfected with itscorresponding putative receptor 300 to 500-fold more than control phage(stippled bar). In addition, phage binding was inhibited in the presenceof 100 μg/ml of cognate peptide (solid bar) but not by unrelated homingpeptides (open bars). These results confirm that the homing peptideCRPPR (SEQ ID NO: 1) specifically binds to the receptor HLP/CRIP2; thehoming peptide CKRAVR (SEQ ID NO: 7) specifically binds to the receptorSigirr/TIR8; the homing peptide CRSTRANPC (SEQ ID NO: 11) specificallybinds to MpcII-3-related protein receptor; and that the homing peptideCPKTRRVPC (SEQ ID NO: 12) specifically binds to the receptor bc10.

Receptor transfections and phage binding assays were performed asfollows. 293T cells were transfected with pMH6-derived plasmids (RocheDiagnostics; Indianapolis, Ind.) encoding HLP/CRIP2, Sigirr/TIR8,MpcII-3-related protein and bc10 using the Fugene transfection reagent(Roche Diagnostics). Briefly, 10 μg of plasmid was mixed with 700 μl ofserum-free DMEM and 30 μl of Fugene transfection reagent and incubatedfor 15 minutes at room temperature before the mixture was added tocells. After forty-eight hours, cells were detached from culture dishesusing EDTA and washed once with PBS. Phage displaying CRPPR (SEQ ID NO:1), CKRAVR (SEQ ID NO: 7), CRSTRANPC (SEQ ID NO: 11) or CPKTRRVPC (SEQID NO: 12) and nonrecombinant control phage (1×10⁹ p.f.u) were incubatedwith transfected cells for two hours at 4° C. Unbound phage were removedby five washes with 1% BSA in PBS. Bound phage were rescued by additionof bacteria; binding efficiencies were determined by plaque assay asdescribed above.

Peptides for competition assays were synthesized at The BurnhamInstitute peptide facility using Fmoc chemistry in a solid-phasesynthesizer prior to purification by HPLC and confirmation of theiridentity by mass spectrometry. Fluorescein-conjugated peptides weresynthesized as described in Wender et al., Proc. Natl. Acad. Sci. USA97:13003-13008 (2000). Competition assays were performed by addition of100 μg peptide to the cells and phage, followed by incubation at 4° C.for two hours. After five washes with 1% PBS, bound phage were rescuedand quantitated by plaque assay.

C. Receptor mRNAs are Present in Heart Endothelia

Expression of heart homing receptors was studied in tissues using realtime PCR and in situ hybridization. Real time-PCR analysis (FIG. 5A)showed that the receptor for peptide SEQ ID NO: 1, HLP/CRIP2, wasstrongly expressed in the heart and at lower levels in lung and brain.Several other tissues including skeletal muscle were negative forHLP/CRIP2 mRNA. The other heart-homing receptors, Sigirr/TIR8,MpcII-3-related protein and bc10, were also most highly expressed in theheart with lower levels in other tissues (FIG. 5A). The specificity ofRT-PCR analysis was confirmed by gel electrophoresis (FIG. 5B) coupledwith melting curves and DNA sequence analysis.

In situ hybridization was used to confirm the RT-PCR analysis anddetermine whether the receptors of interest were expressed onendothelial cells of the heart and other tissues. As shown in FIG. 5C,first row, the HLP/CRIP2 signal was abundant throughout the heart andexhibited a vascular expression pattern (FIG. 5C; first row). Tracelevel expression was also observed in lung and brain but not in kidney.Although in situ hybridization detected HLP/CRIP2 mRNA expression in thelungs, little CRPPR (SEQ ID NO: 1) phage homing was detected in thistissue (see above).

Sigirr/TIR8 in situ hybridization analysis also confirmed the RT-PCRresults. The signal was strong in heart vasculature (FIG. 5, secondrow), weaker in lung and kidney, and absent from the brain.MpcII-3-related protein mRNA was present throughout the heart, mostprominently in heart vessels (FIG. 5, third row). MpcII-3-relatedprotein mRNA was also present in some lung blood vessels, although itwas absent from brain and kidney. As further shown in FIG. 5C, last row,bc10 mRNA was present in heart blood vessels and was further present insome lung, kidney and muscle blood vessels but not in blood vessels ofthe brain. In sum, the in situ hybridization results large confirm theRT-PCR results: the four heart-homing receptors are most abundantlyexpressed in the heart. Furthermore, mRNA for the four heart-homingreceptors is localized at the heart endothelium, consistent withlocalization of the disclosed peptides that selectively home to lungvasculature.

Nonradioactive in situ hybridization was performed essentially asdescribed in St Croix et al., Science 289:1197-1202 (2000). Briefly,digoxigenin (DIG)-labeled antisense RNA probes were generated by PCRamplification using an SP6 promoter incorporated into the antisenseprimer and a T7 promoter incorporated into the sense primer. In vitrotranscription was performed with DIG RNA labeling reagents and T7 RNApolymerase (Roche Diagnostics). Frozen tissue sections were fixed with4% paraformaldehyde, permeabilized with pepsin, and incubated with theRNA probes (200 ng/ml) overnight at 55° C. A horseradish peroxidase(HRP) rabbit anti-DIG antibody (DAKO; Carpinteria, Calif.) was used tocatalyze deposition of Biotin-Tyramide using a GenPoint kit for signalamplification (DAKO). Further amplification was achieved by addinghorseradish peroxidase (HRP) rabbit anti-biotin (DAKO), biotin-tyramide,and alkaline phosphatase (AP) rabbit anti-biotin (DAKO). Signal wasdetected with the AP substrate Fast Red TR/Napthol AS-MX (Sigma) intissue sections counterstained with hematoxylin.

Real time polymerase chain reaction (PCR) assays were performedessentially as described in Galang et al., J. Biol. Chem.279:11281-11292 (2004). Briefly, total RNA was extracted from the organsof interest with the RNeasy kit (Qiagen; Valencia, Calif.), and cDNAprepared from 50 ng of whole RNA using the SuperScript II First-StrandSynthesis System kit (Invitrogen; Carlsbad, Calif.). Real time PCRs wereperformed using the LightCycler SYBR green DNA master mix (RocheDiagnostics) according to the manufacturer's instructions. For relativeexpression analysis, the level of the control gene glyceraldehydephosphate dehydrogenase (GAPDH) was used to normalize receptorexpression. GAPDH-normalized expression in heart was defined for eachreceptor as 100%, and used as the basis of comparison. The specificityof real-time PCR was confirmed by melting curves of each PCR product aswell as agarose gel electrophoresis and DNA sequence analysis.

Example IV Intravenously Injected CRPPR (SEQ ID NO: 1) PeptideCo-Localizes with HLP/CRIP2 Protein in Heart Blood Vessels

This example demonstrates co-localization of peptide CRPPR (SEQ IDNO: 1) with its cognate receptor, HLP/CRIP2.

The localization of CRPPR (SEQ ID NO: 1) peptide was compared with theexpression of HLP/CRIP2 receptor in the heart using an anti-HLP/CRIP2antibody. As shown in FIGS. 6A and 6B, fluorescence from intravenouslyinjected CRPPR (SEQ ID NO: 1) peptide co-localized extensively with thevascular marker CD31 both in heart blood vessels and in the endocardium.Furthermore, a fluorescein-labeled control peptide was not detected inthe heart (FIG. 6C). Anti-HLP/CRIP2 staining also co-localized with CD31and peptide CRPPR (SEQ ID NO: 1) in heart vessels and heart endocardium(FIGS. 6D to 6F).

The specificity of CRPPR (SEQ ID NO: 1)-phage binding to heartvasculature was assayed using competition assays. As shown in FIGS. 7Aand 7B, both CRPPR (SEQ ID NO: 1) peptide and anti-HLP/CRIP2 antibodyblocked binding of the CRPPR (SEQ ID NO: 1)-displaying phage toheart-derived cell suspensions in a dose-dependent manner. As negativecontrols, two other heart-homing peptides had no effect on the CRPPR(SEQ ID NO: 1)-phage binding. Furthermore, co-injected anti-HLP/CRIP2antibody inhibited in vivo homing of CRPPR (SEQ ID NO: 1)-phage (FIG.7C). In sum, these results demonstrate that the peptide CRPPR (SEQ IDNO: 1) selectively homes to heart vasculature through specific bindingto HLP/CRIP2.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. An isolated peptide or peptidomimetic, said isolated peptide selectedfrom the group consisting of a peptide having a length of less than 60residues and comprising the amino acid sequence CRPPR (SEQ ID NO: 1), apeptide comprising the amino acid sequence GRKSKTV (SEQ ID NO: 14), apeptide having a length of less than 150 residues and comprising theamino acid sequence CARPAR (SEQ ID NO: 5), a peptide having a length ofless than 50 residues and comprising the amino acid sequence CPKRPR (SEQID NO: 6), a peptide having a length of less than 400 residues andcomprising the amino acid sequence CKRAVR (SEQ ID NO: 7), a peptidecomprising the amino acid sequence RNSWKPN (SEQ ID NO: 16), a peptidecomprising the amino acid sequence RGSSS (SEQ ID NO: 9), a peptidehaving a length of less than 400 residues and comprising the amino acidsequence RSTRANP (SEQ ID NO: 18), a peptide having a length of less than400 residues and comprising the amino acid sequence PKTRRVP (SEQ ID NO:20), and a peptide having a length of less than 400 residues andcomprising the amino acid sequence SGMARTK (SEQ ID NO: 22), or apeptidomimetic thereof.
 2. The isolated peptide or peptidomimetic ofclaim 1, which is a peptide. 3-11. (canceled)
 12. An isolated peptide orpeptidomimetic, said isolated peptide selected from the group consistingof a peptide comprising the amino acid sequence CXGRKSKTVZC (SEQ ID NO:15), a peptide comprising the amino acid sequence CXRNSWKPNZC (SEQ IDNO: 17), a peptide comprising the amino acid sequence CXRSTRANPZC (SEQID NO: 19), a peptide comprising the amino acid sequence CXPKTRRVPZC(SEQ ID NO: 21), and a peptide comprising the amino acid sequenceCXSGMARTKZC (SEQ ID NO: 23), or a peptidomimetic thereof, wherein X=0 to20 independently selected residues and wherein Z=0 to 20 independentlyselected residues. 13-126. (canceled)
 127. A conjugate, comprising atherapeutic agent linked to a homing molecule that selectively homes toheart vasculature, said homing molecule specifically bindingcysteine-rich protein 2 (HLP/CRIP2; SEQ ID NO: 25), single Ig IL-2receptor related protein (Sigirr/TIR8; SEQ ID NO: 29), SEQ ID NO: 33,murine bladder cancer-associated protein homolog (bc10; SEQ ID NO: 35).128. The conjugate of claim 127, wherein said homing molecule homes tothe heart in vivo with a selectivity of at least 5-fold relative tonon-recombinant phage.
 129. The conjugate of claim 127, wherein saidhoming molecule is a peptide or peptidomimetic. 130-135. (canceled) 136.The conjugate of claim 133, wherein said homing peptide orpeptidomimetic is conformationally constrained.
 137. The conjugate ofclaim 127, wherein said homing molecule is an antibody orantigen-binding fragment thereof.
 138. The conjugate of claim 127wherein said therapeutic agent is selected from the group angiogenicagent, anti-thrombotic agent, anti-inflammatory agent, immunosuppressiveagent, anti-arrhythmic agent, tumor necrosis factor inhibitor,endothelin inhibitor, angiotensin-converting enzyme (ACE) inhibitor,calcium antagonist, antibiotic agent, antiviral agent and viral vector.139. The conjugate of claim 138, wherein said therapeutic agent is anangiogenic agent.
 140. The conjugate of claim 138, wherein saidtherapeutic agent is an anti-thrombotic agent. 141-241. (canceled)